Lateral Flow Quantitative Assay Method and Strip and Laser-Induced Fluorescence Detection Device Therefor

ABSTRACT

Disclosed is a lateral flow quantitative assay method which can measure one or more analyte species at the same time, with high sensitivity. Also, the present invention relates to a strip which can measure one or more analyte species at the same time, with high sensitivity and a package in which the strip is integrated with a laser-induced surface fluorescence detector. The present invention can quantify multiple analytes with a minimum detection limit of pg/ml. Therefore, the present invention provides an advantage capable of quantifying a plurality of analytes at the same time using a simple lateral flow assay strip.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Divisional Application of U.S. patent application Ser. No.10/502,378, filed Jul. 23, 2004 and entitled “Lateral Flow QuantitativeAssay Method and Strip and Laser-induced Fluorescence Detection DeviceTherefor”; and which claims priority of Korean PCT application no.PCT/KR03/00151, entitled “Lateran Flow Quantitative Assay Method andStrip and Laser-Induced Fluorescing Detection Device Therefor”, filed on23 Jan. 2003 which claims priority of Korea patent application number10-2002-0005755, filed Jan. 31, 2002, entitled “Lateral FlowQuantitative Assay Method and Strip and Laser-induced FluorescenceDetection Device Therefor”; and which claims priority of Korean patentapplication number 10-2002-0003995 entitled “Lateral Flow QuantitativeAssay Method and Strip and Laser-induced Fluorescence Detection DeviceTherefor”, filed on 23 Jan. 2002 all of which are hereby incorporated byreference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

The present invention relates to a lateral flow quantitative assaymethod which can measure one or more analyte species at the same time,with high sensitivity. Also, the present invention relates to a stripwhich can measure one or more analyte species at the same time, withhigh sensitivity and a package in which the strip is integrated with alaser-induced epifluorescence detector.

BACKGROUND OF THE INVENTION

Over the past 30 years, development of novel diagnostic apparatuses andmethods which involve quantitative and qualitative analyses of extremelysmall quantities of substances contained in a sample taken for biopsy,such as blood or urine, has actively and rapidly progressed and evennow, is still progressing at a high speed. RIA (RadioimmunologicalAssay) using radioactive isotopes was introduced in the 1950s, and ELISA(Enzyme Linked ImmunoSorbent Assay) was developed and advanced in the1970s and 1980s. The ELISA method is the most popular laboratory testtoday and one of requisite tools for research in medical or life sciencefields. Recently, modified ELISA methods have been developed. Amongthem, for example, there is a method for analyzing a plurality ofanalytes at one time by immobilizing a plurality of antibodies onto a96-well plate.

By typical immunodiagnostic methods, including RIA or ELISA, only onekind of analyte per sample can be quantified, using expensive analyticalmachinery and tools, while performing a multi-step procedure. Therefore,these methods cannot be readily used in a small-scale hospital,emergency room, the home, etc., where such equipments are not provided.In order to make up for this weak point, a convenient diagnostic kitusing immunochromatography has been developed.

Using such diagnostic kit, it is possible to obtain a test result in 15minutes after applying a sample such as whole blood, serum, urine, etc.to the kit. A representative type of immunochromatographic assays is alateral flow assay. A kit for the lateral flow assay has a structurecomprising a sample pad, to which a sample is applied, a releasing padcoated with a detector antibody, a developing membrane (typically,nitrocellulose) or strip, in which components of the sample move atdifferent rates to be individually separated and to undergoantibody-antigen reaction, and an absorption pad which is provided atthe far end of the sample pad to cause the sample to keep moving. Thedetector antibody is fixed onto, for example, colloidal gold particlesto enable the detection. Latex beads or carbon particles may be usedinstead of gold particles. The diagnostic kit for the lateral flow assayis generally designed to detect an analyte in a sandwich configurationcomprising the analyte, the detector antibody, and a capture antibody.Upon applying a liquid sample to the sample pad of the kit, an analytecontained in the sample begins to move from a sample pad. Firstly, theanalyte reacts with a detector antibody releasably adhered to areleasing pad to form an antigen-antibody conjugate, which continues todevelop in this conjugated form. Then, while moving through thedeveloping membrane, the antigen-antibody conjugate reacts once morewith a capture antibody fixed on a developing membrane to form a captureantibody-antigen-detector antibody conjugate in a sandwich form. Sincethe capture antibody is fixed on the developing membrane, conjugates areaccumulated in the area where the capture antibodies are fixed. Proteinsare invisible to the naked eye. Therefore, the presence and amount ofconjugates are determined by means of an amount of gold particlesattached to a certain area of the developing membrane.

The lateral flow assay can be widely and conveniently used in variousfields such as pregnancy diagnosis, cancer diagnosis, and microbedetection. However, since quantification cannot be performed with thenaked eye and hence, an exact amount of an analyte cannot be determined,its application is restricted. Especially, when a judgment should bemade around a cut-off value, it is difficult to make an exact diagnosis.For example, in case of prostate cancer, when a detected value is 3.9ng/ml which is very close to the standard cut-off value of 4 ng/ml, anexact diagnosis cannot be made.

Immunodiagnosis is now rapidly developing, and in the near future, willbe able to easily and promptly identify and analyze a sample anddiagnose disease conditions. The RIA or ELISA method which can quantifyan analyte at present involves several complicated steps for suchquantification, including treatment with an enzyme and washing.Similarly, the conventional convenient diagnostic kits have difficultiesin providing quantified results. Therefore, there is a great demand fora general assay method which can perform quantification more rapidly,conveniently and sensitively. With the method, an ordinary unskilledperson can practice diagnosis or analysis in any place.

The conventional lateral flow quantitative assay strips, including thosedisclosed in documents or products commercially available in the market,have a low sensitivity and are now used as means for performing aqualitative assay rather than a quantitative assay of analytes.Recently, in order to examine a disease state, several tens of analytesare generally analyzed, and numbers of analytes needed to be examinedare tending to increase due to the rapid advance of molecular biologyand medical science. However, at the present time, the individualanalytes should be assayed separately, thereby increasing the burden oftime and cost. Under the present circumstances, it would be advantageousin terms of economic aspects and other aspects to provide a methodcapable of rapidly and precisely quantifying different kinds of analytesat the same time, to satisfy demands of both general consumers and thoseinvolved in medical fields for development of such products.

SUMMARY OF THE INVENTION

The present inventors have developed a lateral flow quantitative assaymethod which is capable of quantifying a plurality of analytes at thesame time with a minimum detection limit of pg/ml and a strip therefor,and a package comprising the strip and a laser-induced epifluorescencedetector.

In accordance with an aspect, the present invention provides a lateralflow quantitative assay method in which a liquid sample which isexpected to contain analytes is applied at one end of a chromatographymedium to move through the chromatography medium, such that the analytesin the sample reacts with a labeled detector adsorbed on a sectionlocated at a predetermined distance from the sample application in thesample developing direction, thereby forming an analyte/labeled detectorconjugate; in which the analyte/labeled detector conjugate, while movingthrough the chromatography medium, further reacts with an unlabeledcaptor which is different from or identical to the detector and isimmobilized on a viewing window defined around middle portion of thechromatography medium, thereby forming a labeleddetector/analyte/unlabeled captor conjugate in a sandwich configuration;and in which an amount of the conjugate is measured for quantitativedetermination of the analyte in the sample, characterized in that:

(a) the labeled detector is labeled with fluorescent material and reactswith the analytes in the liquid sample to form a fluorescently-labeleddetector/analyte conjugate;

(b) the unlabeled captor is immobilized in lines within the viewingwindow on the chromatography medium and reacts with thefluorescently-labeled detector/analyte conjugate which has moved alongthe chromatography medium to form a fluorescently-labeleddetector/analyte/unlabeled captor conjugate;

(c) a reference detector which is different from the detector andcaptor, is labeled with the same fluorescent material as the detectorand reacts with reference material in the liquid sample, is adsorbed onthe section of the chromatography medium where the fluorescently-labeleddetector is adsorbed, and an unlabeled reference captor which reactswith the fluorescently-labeled reference detector is immobilized in asingle reference line before or after the viewing window on thechromatography medium, or in double reference lines before and after theviewing window on the chromatography medium, whereby a referenceconjugate of fluorescently-labeled reference detector/referencematerial/unlabeled reference captor is formed as the liquid samplepasses through the chromatography medium; and

(d) an amount of the analytes is determined by passing light emittedfrom a laser through an exciter filter, irradiating the filtered lightto the epifluorescence medium containing the analyte conjugate and thereference conjugate, focusing light reflected from the epifluorescencemedium to a first focal point of an elliptical or spherical reflectingmirror with a proper size, focusing scattered incident light andfluorescence emitted from the sample positioned at the first focal pointof the elliptical reflecting mirror to a second focal point of theelliptical reflecting mirror, converting the focused light into parallellight by a collimator, filtering the parallel light through afluorescent filter to remove the scattered incident light and provide apure a fluorescence component to an optical detector, and comparing afluorescence intensity of the analyte conjugate with a referencefluorescence intensity of the reference conjugate to determine therelative amount of the analyte.

In accordance with a second aspect, the present invention provides alateral flow quantitative assay strip which comprises a backing, asample pad adhered to one end of the backing and to which a liquidsample is applied, a conjugate releasing pad adhered to the backing suchthat one end of the sample pad overlaps with the end of the conjugatereleasing pad closest to the end of the strip to which a sample isapplied and upon which a labeled detector is releasably attached toreact with an analyte in the liquid sample to form a conjugate, achromatography medium adhered to the backing such that one end of themedium is overlapped by the end of the conjugate releasing pad farthestfrom the end of the strip to which the sample is applied and along whichthe sample develops and upon which a captor which is different from oridentical to the detector and reacts with and traps the conjugatereleased from the conjugate releasing pad as the sample develops to forma sandwich type conjugate, and an absorption pad to absorb the sampledeveloping along the chromatography medium and to absorb and removeunreacted labeled substances, characterized in that:

the detector releasably attached to the conjugate releasing pad islabeled with fluorescent material;

a reference detector which is labeled with the same fluorescent materialas that of the detector and reacts with reference material in the liquidsample is further releasably attached to the conjugate releasing pad;

the captor is immobilized in lines within a viewing window on thechromatography medium,

an unlabeled reference captor which is different from the detector andcaptor is immobilized in a single reference line before or after theviewing window on the chromatography medium, or in double referencelines before and after the viewing window on the chromatography medium,whereby a conjugate of fluorescently-labeled detector/analyte/unlabeledcaptor and a reference conjugate of fluorescently-labeled referencedetector/reference material/unlabeled reference captor are formed as theliquid sample passes through the chromatography medium; and

an amount of the analytes is determined by passing light emitted from alaser through an exciter filter, irradiating the filtered light to theepifluorescence medium containing the analyte conjugate and thereference conjugate, focusing light reflected from the epifluorescencemedium to a first focal point of an elliptical or spherical reflectingmirror, focusing scattered incident light and fluorescence emitted fromthe sample positioned at the first focal point of the ellipticalreflecting mirror to a second focal point of the elliptical reflectingmirror, converting the focused light into parallel light by acollimator, filtering the parallel light through a fluorescent filter toremove the scattered incident light and provide a pure fluorescencecomponent to an optical detector, and comparing a fluorescence intensityof the analyte conjugate with a reference fluorescence intensity of thereference conjugate to determine the relative amount of the analyte.

In accordance with a third aspect, the present invention provides alaser-induced epifluorescence detecting method which comprises steps of:passing light emitted from a laser through an exciter filter andfocusing the filtered light to the surface of a sample positioned at afirst focal point of an elliptical or spherical reflecting mirror with aproper size; focusing scattered incident light and fluorescence emittedfrom the sample positioned at the first focal point of the ellipticalreflecting mirror to a second focal point of the elliptical reflectingmirror by reflection of the reflecting mirror; converting the focusedlight into parallel light by a collimator; filtering the parallel lightthrough a fluorescent filter to remove the scattered incident light; andproviding a pure a fluorescence component to an optical detector.

In accordance with a fourth aspect, the present invention provides alaser-induced epifluorescence detecting method which comprises steps of:passing light emitted from a laser through an exciter filter andfocusing the filtered light to the surface of a sample positioned at afirst focal point of an elliptical or spherical reflecting mirror with aproper size; focusing scattered incident light and fluorescence emittedfrom the sample positioned at the first focal point of the ellipticalreflecting mirror to a spatial filter positioned at a second focal pointof the elliptical reflecting mirror by reflection of the reflectingmirror; converting the filtered light into parallel light by acollimator; filtering the parallel light through a fluorescent filter toremove the scattered incident light; and providing a pure a fluorescencecomponent to an optical detector.

In accordance with a fifth aspect, the present invention provides alaser-induced epifluorescence detecting apparatus comprising a laser, anexciter filter, an elliptical reflecting mirror or spherical mirror,epifluorescent sample control means, a collimator, a fluorescent filterand an optical detector, characterized in that the components of thedetecting apparatus are arranged in a structure such that light emittedfrom a laser is passed through an exciter filter and is focused to thesurface of a sample positioned at a first focal point of an ellipticalreflecting mirror with a proper size, scattered incident light andfluorescence emitted from the sample positioned at the first focal pointof the elliptical reflecting mirror are reflected from the ellipticalreflecting mirror and focused upon a collimator as a second focal pointof the elliptical reflecting mirror, by which the focused light isconverted into parallel light and passed through a fluorescent filter toremove the scattered incident light, thereby providing a purefluorescence component to an optical detector.

In accordance with a sixth aspect, the present invention provides alaser-induced epifluorescence detecting apparatus comprising a laser, anexciter filter, an elliptical reflecting mirror or spherical mirror,epifluorescent sample control means, a spatial filter, a collimator, afluorescent filter and an optical detector, characterized in that thecomponents of the detecting apparatus are arranged in a structure suchthat light emitted from a laser is passed through an exciter filter andis focused to the surface of a sample positioned at a first focal pointof an elliptical reflecting mirror with a proper size, scatteredincident light and fluorescence emitted from the sample positioned atthe first focal point of the elliptical reflecting mirror are reflectedfrom the elliptical reflecting mirror and focused to a spatial filter asa second focal point of the elliptical reflecting mirror, and filteredlight is focused upon a collimator, by which the focused light isconverted into parallel light and passed through a fluorescent filter toremove the scattered incident light, thereby providing a purefluorescence component to an optical detector.

In accordance with a seventh aspect, the present invention provides apackage for quantitative assay comprising (i) a lateral flowquantitative assay strip comprising a backing, a sample pad adhered toone end of the backing and to which a liquid sample is applied, aconjugate releasing pad adhered to the backing such that one end of thesample pad overlaps with the end of the conjugate releasing pad closestto the end of the strip to which the sample is applied and upon which alabeled detector is releasably attached to react with an analyte in theliquid sample to form a conjugate, a chromatography medium adhered tothe backing such that one end of the medium is overlapped by the end ofthe conjugate releasing pad farthest from the end of the strip to whichthe sample is applied and along which the sample develops and upon whicha captor which is different from or identical to the detector and reactswith and traps the conjugate released from the conjugate releasing padas the sample develops to form a sandwich type conjugate, and anabsorption pad to absorb the sample developing along the chromatographymedium and to absorb and remove unreacted labeled substances; in whichthe detector releasably attached to the conjugate releasing pad islabeled with fluorescent material, a reference detector which is labeledwith the same fluorescent material as that of the detector and reactswith reference material in the liquid sample is further releasablyattached to the conjugate releasing pad, the captor is immobilized in aline within a viewing window on the chromatography medium, an unlabeledreference captor which is different from the detector and captor isimmobilized in a single reference line before or after the viewingwindow on the chromatography medium, or in double reference lines beforeand after the viewing window on the chromatography medium, and (ii) alaser-induced epifluorescence detecting apparatus comprising a laser, anexciter filter, an elliptical reflecting mirror or spherical mirror,epifluorescence sample control means, a collimator, a fluorescent filterand an optical detector, characterized in that:

the components of the detecting apparatus are arranged in a structuresuch that light emitted from a laser is passed through an exciter filterand irradiated to the epifluorescence medium containing the analyteconjugate of fluorescently-labeled detector/analyte/unlabeled captorformed in the viewing window and the reference conjugate offluorescently-labeled reference detector/reference material/unlabeledreference captor formed in the reference lines as the liquid samplepasses through the chromatography medium of the strip, light reflectedfrom the surface is focused to a first focal point of an elliptical orspherical reflecting mirror with a proper sixe, scattered incident lightand fluorescence emitted from the sample positioned at the first focalpoint of the elliptical reflecting mirror are reflected from theelliptical reflecting mirror and focused to a collimator as a secondfocal point of the elliptical reflecting mirror, by which the focusedlight is converted into parallel light and passed through a fluorescentfilter to remove the scattered incident light, thereby providing a purefluorescence component to an optical detector, in which a fluorescenceintensity of the conjugate and a reference fluorescence intensity of thereference conjugate, each being formed on the strip, are measured, andwhich is capable of determining the quantity of the analyte in theliquid sample by comparing the fluorescence intensities of theconjugates measured by the laser-induced epifluorescence detectingapparatus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the conventional lateral flowquantitative assay strip.

FIG. 2 is a perspective view of the lateral flow quantitative assaystrip of an embodiment according to the present invention.

FIG. 3 is a plan view of the conventional lateral flow quantitativeassay strip shown in FIG. 1.

FIG. 4 is a plan view of the lateral flow quantitative assay strip ofthe embodiment according to the present invention shown in FIG. 2.

FIG. 5 is a side view of the lateral flow quantitative assay strip ofthe embodiment according to the present invention shown in FIG. 2 andFIG. 4.

FIG. 6 is a side view of a lateral flow quantitative assay strip ofanother embodiment according to the present invention.

FIG. 7 is a side view of a lateral flow quantitative assay strip ofanother embodiment according to the present invention.

FIG. 8 is a side view of a lateral flow quantitative assay strip ofanother embodiment according to the present invention.

FIG. 9 is a side view of a lateral flow quantitative assay strip ofanother embodiment according to the present invention.

FIG. 10 is a side view of a lateral flow quantitative assay strip ofanother embodiment according to the present invention.

FIG. 11 is a side view of a lateral flow quantitative assay strip ofanother embodiment according to the present invention.

FIG. 12 is a view illustrating a structure of a laser-inducedepifluorescence detecting apparatus with an elliptical reflecting mirroraccording to the present invention.

FIG. 13 is a view illustrating a structure of a laser-inducedepifluorescence detecting apparatus with a spherical reflecting mirroraccording to the present invention.

FIG. 14 is a graph showing the minimum detection limit concentration ofa surface antigen of an analyte, as measured using a laser-inducedepifluorescence detecting apparatus with an elliptical reflecting mirroraccording to the present invention (A: 4 ng/ml; B: 400 pg/ml; C: 40pg/ml).

FIG. 15 is a graph showing epifluorescence intensities of PSA (ProstateSpecific Antigen), as measured using a laser-induced epifluorescencedetecting apparatus with an elliptical reflecting mirror (A) accordingto the present invention and a conventional fluorescence detectingscanner (B).

FIG. 16 is a graph showing results of quantification of a multi-testline strip using a laser-induced epifluorescence detecting apparatuswith an elliptical reflecting mirror according to the present invention.

FIG. 17 is a schematic view of the bound reactants in the test lineregion on the strip when using the biotin-avidin system according to thepresent invention and the conventional method.

FIG. 18 is a graph showing the results of quantification using thebiotin-avidin system according to the present invention and theconventional method.

FIG. 19 is a graph showing the result of measuring the fluorescenceintensity of the total PSA using the laser-induced epifluorescencedetecting apparatus according to the present invention.

FIG. 20 is a graph showing the result of measuring the fluorescenceintensity of the free PSA using the laser-induced epifluorescencedetecting apparatus according to the present invention.

FIG. 21 is a view illustrating a structure of a conventionallaser-induced epifluorescence detector.

FIG. 22 is a graph showing results of quantification of a protein chipwhere antibody proteins are arranged in one dimension using alaser-induced epifluorescence detecting apparatus with an ellipticalreflecting mirror according to the present invention.

FIG. 23 is a view illustrating a structure of a sample controller, acomponent of the laser-induced epifluorescence detecting apparatusaccording to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “sensitivity” as used herein refers to a minimum quantity of aconjugate of a captor, detector and analyte which can be detected.

The term “epifluorescence” as used herein refers to the fluorescenceemitted from a conjugate of fluorescently-labeleddetector/analyte/captor and/or a reference conjugate offluorescently-labeled reference detector/reference material/referencecaptor, which are fixed in a viewing window and a reference line,respectively, of the lateral flow assay strip by chromatography.

The term “analyte” as used here in refers to a compound or compositionbeing analyzed in a liquid sample. The samples which are usable in thepresent invention may be selected from any samples containing such ananalyte. Examples include physiological fluid such as urine, serum,plasma, blood, saliva, spinal fluid, ocular liquid, amniotic fluid,etc., food such as milk and wine, chemical treatment stream such asdomestic waste water. Analytes that can be examined in the presentinvention are largely classified into a complete antigen and a hapten(incomplete antigen). The complete antigen refers to an antigenicsubstance which itself has the ability to induce antibody production(immunogenicity), and mainly includes peptide hormones having highmolecular weights. The hapten (incomplete antigen) refers a materialwhich can bind to an antibody but has no ability to induce antibodyproduction by itself, and includes peptides having relatively lowmolecular weights (molecular weights of about 1,000 or less). Haptensacquire the ability to induce antibody production when bound to aprotein such as bovine serum albumin.

For the purposes of the present invention, examples of the completeantigens are described below, but are not limited thereto:

(1) Examples of Peptide Hormones

-   -   1) Pituitary hormones such as growth hormone (GH),        adrenocorticotropic hormone (ACTH), melanocyte-stimulating        hormone (MSH), prolactin, thyroid-stimulating hormone (TSH),        luteinizing hormone (LH), follicle-stimulating hormone (FSH) and        oxytocin;    -   2) Calcium metabolic regulatory hormones such as calcitonin and        parathyroid hormone;    -   3) Insulin, proinsulin and pancreatic hormone;    -   4) Alimentary canal hormones such as gastrin and secretin;    -   5) Hormones which act on blood vessels such as angiotensin and        bradykinin; and    -   6) Placental hormones such as human chorionic gonadotropin (hCG)        and human placental lactogen (hPL).

(2) Examples of Other Substances

-   -   1) Enzymes such as prostatic acidic phosphatase (PAP),        prostate-specific antigen (PSA), alkaline phosphatase,        transaminase, lactic acid dehydrogenase (LDH), transaminase,        trypsin and pepsinogen;    -   2) Cancer-specific substances such as α-fetoprotein (AFP) and        cancer embryonic antigen (CEA);    -   3) Serum protein components such as immunoglobulin G (IgG),        fibrin-fibrinogen decomposition products (FDP, D-dimer),        antithrombin III (ATIII) and transferrin; and    -   4) Substances such as rheumatoid factor, serotonin, urokinase,        ferritin and substance P.

For the purposes of the present invention, examples of haptens aredescribed below, but are not limited thereto:

(1) Steroidal Haptens

-   -   1) Estrogens such as estrone, estradiol, estriol, estetrol,        equilin and equilenin;    -   2) Natural or synthetic luteohormones such as progesterone,        pregnanediol, pregnanetriol, 19-norethisterone and        chloromadinone acetate;    -   3) Male sex hormones such as testosterone,        dehydroepiandrosterone, dihydrotestosterone, androsterone and        etiocholanorone;    -   4) Adrenal cortical hormones such as cortisol, cortisone,        deoxycorticosterone, aldosterone and tetrahydrocortisol; and    -   5) Bile acids such as vitamins D, cholesterol, cholic acid,        deoxycholic acid and chenocholic acid, and other steroids such        as cardiotonic steroid, saponin and sapogenin.

(2) Physiologically active amines

-   -   1) Catecholamines such as epinephrine, norepinephrine, dopamine        and ephedrine, and metabolites thereof;    -   2) Physiologically active alkaloids such as morphine, codeine,        heroin, morphine chloride, cocaine, mescaline, papaverine,        narcotine, yohimbine, reserpine, ergotamine and strychnine; and    -   3) Amino group-containing psychotropics such as LSD,        amphetamine, methamphetamine and meprobamate.

(3) Other Examples

-   -   1) Low-molecular-weight peptides having no antigenicity such as        TRH and LH-RH;    -   2) Thyroid hormones such as diiodothyronine, triiodothyronine        and thyroxine;    -   3) Prostaglandins such as prostaglandin E2, prostaglandin E3 and        prostaglandin F1a;    -   4) Vitamins such as vitamin A, B vitamins (vitamins B1, B2, B6        and B12, and the like), vitamin E and vitamin K;    -   5) Antibiotics such as penicillin, actinomycin, chloromycetin        and tetracycline; and    -   6) Other in vivo components, and drugs administered into        organisms and metabolites thereof.

According to the present invention, the analytes are characterized bybeing monoepitopic or polyepitopic. The polyepitopic ligand analyteswill normally be poly(amino acids) i.e. polypeptides and proteins,polysaccharides, nucleic acids, and combinations thereof. Suchcombinations of assemblages include bacteria, viruses, chromosomes,genes, mitochondria, nuclei, cell membranes, and the like. For the mostpart, the polyepitopic ligand analytes employed in the present inventionwill have a molecular weight of at least about 5,000, more usually atleast about 10,000. In the poly(amino acid) category, the poly(aminoacids) of interest will generally be from about 5,000 to 5,000,000 inmolecular weight, more usually from about 20,000 to 1,000,000 inmolecular weight, and among the hormones of interest, the molecularweights will usually range from about 5,000 to 60,000.

The wide variety of proteins may be classified into families of proteinshaving similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc. For cells and viruses,histocompatability antigens or surface antigens will frequently be ofinterest.

The proteins related by structure are classified into protamines,histones, albumins, globulins, scleroproteins, phosphoproteins,mucoproteins, chromoproteins, lipoproteins, nucleoproteins,glycoproteins, and proteoglycans. In addition, unclassified proteins,for example, somatotropin, prolactin, insulin, pepsin and the like maybe included. All of these proteins can be quantified by the packagecomprising the lateral flow assay strip and the laser-inducedepifluorescence detecting apparatus according to the present invention.

A number of proteins found in human plasma which are clinicallyimportant can also be quantified by the package comprising the lateralflow assay strip and the laser-induced epifluorescence detectingapparatus according to the present invention. Examples of such plasmaproteins include prealbumin, albumin, α₁-lipoprotein, α₁-acidglycoprotein, α₁-antitrypsin, α₁-glycoprotein, transcortin,4.6S-postalbumin, tryptophan-poor α₁-glycoprotein, α₁X-glycoprotein,thyroxin-binding globulin, inter-α-trypsin-inhibitor, Gc-globulin (Gc1-1, Gc 2-1 and Gc 2-2), haptoglobin (Hp 1-1, Hp 2-1 and Hp 2-2),ceruloplasmin, cholinesterase, α₂-lipoprotein(s), myoglobin, C-reactiveprotein, α₂-macroglobulin, α₂-HS-glycoprotein, Zn-α₂-glycoprotein,α₂-neuramino-glycoprotein, erythropoietin, β-lipoprotein, transferrin,hemopexin, fibrinogen, plasminogen, β₂-glycoprotein I, β₂-glycoproteinII, immunoglobulin G (IgG), A (IgA), M (IgM), D (IgD), E (IgE) and thelike.

Other examples of analytes which can be quantified by the packagecomprising the lateral flow assay strip and the laser-inducedepifluorescence detecting apparatus according to the present inventionare complement factors and blood clotting factors. Examples of thecomplement factors include C′1, C′1q, C′1r, C′1s, C′2, C′3 (β₁A andα₂D), C′4, C′5, C′6, C′7, C′8 and C′9. Important blood clotting factorsinclude fibrinogen, prothrombin, thrombin, tissue thromboplastin,proaccelerin, globulin (accelerator of proaccelerin), proconvertin,antihemophilic globulin (AHG), Christmas factor (plasma thromboplastincomponent (PTC)), Stuart-Prower factor (autoprothrombin III), plasmathromboplastin antecedent (PTA), Hagemann factor and fibrin-stabilizingfactor.

Important protein hormones which can be quantified by the packageaccording to the present invention include, but are not limited to,peptide and protein hormones such as parathyroid hormone (parathromone),thyrocalcitonin, insulin, glucagons, relaxin, erythropoietin,melanotropin, somatotropin (growth hormone), corticotropin, thyrotropin,follicle-stimulating hormone, luteinizing hormone, luteomammotropichormone and gonadotropin (chorionic gonadotropin); tissue hormones suchas secretin, gastrin, angiotensin I and II, bradykinin and humanplacental lactogen; peptide hormones from the neurohypophysis such asoxytocin, vasopressin, and releasing factors (RF) (CRF, LRF, TRF,somatotropin-RF, GRF, FSH-RF, PIF, MIF).

Still other analytes which can be quantified by the package according tothe present invention include antigenic polysaccharides derived frommicroorganisms. Examples of the antigenic polysaccharides derived frommicroorganisms include, but are not limited to hemosensitins found inStreptococcus pyogenes polysaccharide, Diplococcus pneumoniaepolysaccharide, Neisseria meningitidis polysaccharide, Neisseriagonorrheae polysaccharide, Corynebacterium diphtheriae polysaccharide,Actinobacillus mallei crude extract, Francisella tularensislipopolysaccharide and polysaccharide, Pasteurella pestispolysaccharide, Pasteurella multocida capsular antigen, Brucella abortuscrude extract, Haemophilus influenzae polysaccharide, Haemophiluspertussis crude extract, Treponema reiteri polysaccharide, Veillonellalipopolysaccharide, Erysipelothrix polysaccharide, Listeriamonocytogenes polysaccharide, Chromobacterium lipopolysaccharide,Mycobacterium tuberculosis saline extract of 90% phenol-extractedmycobacteria and polysaccharide fraction, Klebsiella aerogenespolysaccharide, Klebsiella cloacae polysaccharide, Salmonella typhosaliposaccharide and polysaccharide, Salmonella typhimuriumpolysaccharide, Shigella dysenteriae polysaccharide, Shigella flexneriand Shigella sonnei crude extract and polysaccharide, Rickettsiae crudeextract, Candida albicans polysaccharide and Entamoeba histolytica crudeextract.

The microorganisms which are assayed using the package according to thepresent invention may be intact, lysed, ground or otherwise fragmented.Examples of such microorganisms include Corynebacteria, Corynebacteriumdiptheriae, Pneumococci, Diplococcus pneumoniae, Streptococci,Streptococcus pyogenes, Streptococcus salivarus, Staphylococci,Staphylococcus aureus, Staphylococcus albus, Neisseriae, Neisseriameningitides, Neisseria gonorrheae, Enterobacteriaciae, Escherichiacoli, Aerobacter aerogenes, Klebsiella pneumoniae bacteriam, Salmonellatyphosa, Salmonella choleraesuis, Salmonella typhimurium, Shigelladysenteriae, Shigella schmitzii, Shigella arabinotarda, Shigellaflexneri, Shigella boydii, Shigella Sonnei, Proteus vulgaris, Proteusmirabilis, Proteus morgani, Pseudomonas aeruginosa, Alcaligenesfaecalis, Vibrio cholerae, Hemophilus influenzae, Hemophilus ducreyi,Hemophilus hemophilus, Hemophilus aegypticus, Hemophilus parainfluenzae,Bordetella pertussis, Pasteurellae, Pasteurella pestis, Pasteurellatulareusis, Brucellae, Brucella melitensis, Brucella abortus, Brucellasuis, Bacillus anthracis, Bacillus subtilis, Bacillus megaterium,Bacillus cereus, Clostridium tetani, Clostridium perfringens,Clostridium novyi, Clostridium septicum, Clostridium histolyticum,Clostridium tertium, Clostridium bifermentans, Clostridium sporogenes,Mycobacteria, Mycobacterium tuberculosis hominis, Mycobacterium bovis,Mycobacterium avium, Mycobacterium leprae, Mycobacteriumparatuberculosis, Actinomyces israelii, Actinomyces bovis, Actinomycesnaeslundii, Nocardia asteroids, Nocardia brasiliensis, Spirochetes,Treponema pallidum Spirillum minus, Treponema pertenue Streptobacillus,Treponema carateum, Borrelia recurrentis, Leptospira icterohemorrhagiae,Leptospira canicola, Mycoplasmas, Mycoplasma pneumoniae, Listeriamonocytogenes, Erysipelothrix rhusiopathiae, Streptobacillusmoniliformis, Donvania granulomatis, Bartonella bacilliformis,Rickettsia prowazekii, Rickettsia mooseri, Rickettsia rickettsii,Rickettsia conori, Rickettsia australis, Rickettsia sibiricus,Rickettsia akari, Rickettsia tsutsugamushi, Rickettsia burnetii,Rickettsia Quintana, Chlamydia, Cryptococcus neoformans, Blastomycesdermatidis, Histoplasma capsulatum, Coccidioides immitis,Paracoccidioides brasiliensis, Candida albicans, Aspergillus fumigatus,Mucor corymbifer (Absidia corymbifera), Rhizopus oryzae, Rhizopusarrhizus, Rhizopus nigricans, Sporotrichum schenkii, Fonsecaea pedrosoi,Fonsecaea compacta, Fonsecae dermatidis, Cladosporium carrionii,Phialophora verrucosa, Aspergillus nidulans, Madurella mycetomi,Madurella grisea, Allescheria boydii, Phialosphora jeansilmei,Microsporum gypseum, Trichophyton mentagrophytes, Keratinomyces ajelloi,Microsporum canis, Trichophyton rubrum, Microsporum adnouini,Adenoviruses, Herpes Viruses, Herpes simplex, Varicella, Herpes Zoster,Cytomegalovirus, Pox Viruses, Variola, Vaccinia, Poxvirus bovis,Paravaccinia, Molluscum contagiosum, Picaornaviruses, Poliovirus,Coxsackievirus, Echoviruses, Rhinoviruses, Myxoviruses, Influenza (A, B,and C), Parainfluenza (1-4), Mumps Virus, Newcastle Disease Virus,Measles Virus, Rinderpest Virus, Canine Distemper Virus, RespiratorySyncytial Virus, Rubella Virus, Arboviruses, Eastern Equine EucephalitisVirus, Western Equine Eucephalitis virus, Sindbis Virus, ChikugunyaVirus, Semliki Forest Virus, Mayora Virus, St. Louis Encephalitis Virus,California Encephalitis Virus, Colorado Tick Fever Virus, Yellow FeverVirus, Dengue Virus, Reovirus Types 1-3, Hepatitis A Virus, Hepatitis BVirus, Tumor Viruses, Rauscher Leukemia Virus, Gross Virus, MaloneyLeukemia Virus, Epstein Barr Virus, and other parasites related todiseases such as Dog Heart Worm (microfilaria), Malaria,Schistosomiasis, Coccidosis and Trichinosis.

The monoepitopic ligand analytes which can be quantified using thepackage of the present invention will generally have a molecular weightfrom about 100 to 2,000, more usually from 125 to 1,000. Representativeexamples of the analytes include drugs, metabolites, pesticides,pollutants, and the like. Included among drugs are the alkaloids. Amongthe alkaloids are morphine alkaloids, for example morphine, codeine,heroin, dextromethorphan, their derivatives and metabolites; cocainealkaloids, for example cocaine and benzoyl ecgonine, their derivativesand metabolites; ergot alkaloids, for example the diethylamide oflysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazolinealkaloids, insoquinoline alkaloids; quinoline alkaloids, for examplequinine and quinidine; diterpene alkaloids; and their derivatives andmetabolites.

Also, drugs of steroids can be quantified by the package of the presentinvention. Specific examples thereof include estrogens, gestogens,androgens, andrenocortical steroids, bile acids, cardiotonic glycosidesand aglycones, for example digoxin and digoxigenin, saponins andsapogenins, their derivatives and metabolites. Further included are thesteroid mimetic substances, such as diethylstilbestrol. Another group ofdrugs which can be quantified by the package of the present invention islactams having from 5 to 6 annular members, which include thebarbiturates, for example, phenobarbital and secobarbital,diphenylhydantonin, primidone, ethosuximide, and metabolites thereof.The next group of drugs is aminoalkylbenzenes, in which the alkyl grouphas from 2 to 3 carbon atoms. Examples include the amphetamines,catecholamines such as ephedrine, L-dopa, epinephrine, narceine,papaverine, and metabolites thereof. The next group of drugs isbenzheterocyclics, for example oxazepam, chlorpromazine, tegretol,imipramine, and derivatives and metabolites thereof, in which theheterocyclic rings are azepines, diazepines and phenothiazines. The nextgroup of drugs is purines, for example theophylline, caffeine, andmetabolites and derivatives thereof. The next group of drugs includesthose derived from marijuana, for example cannabinol andtetrahydrocannabinol. The next group of drugs includes the vitamins suchas A, B, for example B₁₂, C, D, E and K, folic acid, and thiamine. Thenext group of drugs is prostaglandins, which differ by the degree andsites of hydroxylation and unsaturation. The next group of drugs isantibiotics, for example, penicillin, chloromycetin, actinomycetin,tetracycline, terramycin, and metabolites and derivatives thereof. Thenext group of drugs is the nucleosides and nucleotides, for example,ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidine with theirappropriate sugar and phosphate substituents. The next group of drugs ismiscellaneous individual drugs, for example, methadone, meprobamate,serotonin, meperidine, amitriptyline, nortriptyline, lidocaine,procaineamide, acetylprocaineamide, propranolol, griseofulvin, valproicacid, butyrophenones, antihistamines, anticholinergic drugs, such asatropine, and metabolites and derivatives thereof. Metabolites relatedto conditions of disease include spermine, galactose, phenylpyruvic acidand porphyrin Type 1. The next group of drugs is aminoglycosides, suchas gentamicin, kanamicin, tobramycin, and amikacin.

The analytes which can be quantified by the package of the presentinvention also include pesticides. Their examples are polyhalogenatedbiphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenatedsulfenamides, metabolites and derivatives thereof.

The analytes which can be quantified by the package of the presentinvention further include receptor analytes, whose molecular weightswill generally range from 10,000 to 2×10⁸, more usually from 10,000 to10⁶. For immunoglobulins, IgA, IgG, IgE and IgM, the molecular weightswill generally vary from about 160,000 to about 10⁶. Enzymes willnormally range from about 10,000 to 6,000,000 in molecular weight.Natural receptors vary widely, generally being at least about 25,000molecular weight and may have a molecular weight of 10⁶ or higher,including such materials as avidin, thyroxine binding globulin,thyroxine binding prealbumin, transcortin, etc.

In addition to the above-described analytes, the package of the presentinvention may be used to quantify tumor markers, angiogenesis relatedmarkers, cardiac markers, Alzheimer disease related markers, cancerrelated genes, environmental toxins, abused drugs and the like. Asexamples of the tumor markers, alpha 1-acidglycoprotein, CEA, AFP,PSA/free PSA, CA 15-3, CA 19-9, CA 27-9, CA-50, CA 125, CA 724,calcitonin, elastase-1, ferritin, pepsinogen I, PIVKA II, ProcollagenIII peptide, beta HCG, beta 2-microglobulin, neuron specific enolase,CYFRA 21-1 (Cytokeratin 19), Secretin, NMP (nuclear matrix protein),COX-1, TPA (Tissue Polypeptide, Antigen) and the like may be included.The angiogenesis related markers include angiogenic factors andangiostatic factors. Specific examples of the angiogenic factors includeaFGF (acidic Fibroblast Growth Factor), bFGF (basic Fibroblast GrowthFactor), VEGF (Vascular Endothelial Growth Factor), angiogenin,angiopoietin 1, heparinase, scatter factor, HGF (Hepatocyte GrowthFactor), PDGF (Platelet Derived Growth Factor), Pleiotrophin, TGF α, TFGβ, IL-8, TNF α, and prostagladins E1 and E2. Specific examples of theangiostatic factors include endostatin, angiostatin, cartilage-derivedinhibitor, heparinase, angiopoietin2, IFN α, IFN β, IFN γ plateletfactor 4, 16 kDa prolactin fragment, protamine, thrombospandin, TIMPs(Tissue Inhibitor of Metalloproteinase), thalidomide and TNP 470(Fumagilin analogue). Examples of the cardiac markers include creatinkinase-BB, creatin kinase-MB, creatin kinase-MM, myoglobin, MLC (MyosinLight Chain), troponin 1, troponin C, troponin ITC, troponin T, CRP andFABP (Fatty Acid Binding Protein). Examples of the Alzheimer diseaserelated markers include glutamine synthetase, melano transferrin andβ-amyloid protein. Examples of the cancer related gene include bcl-2,C-erbB-2, C-myc, CSF-1 receptor, EGF receptor, H-ras, K-ras (p12),L-myc, mdr-1, N-myc, N-ras, p53 exon 4, p53 exon 5, p53 exon 6, p53 exon7, p53 exon 8, p53 exon 9, TcR-α, TcR-β, TcR-γ and TcR-δ. Theenvironmental toxins include for example, microcystin, dioxin and PCB.Examples of the abused drugs include amphetamines, barbiturates,benzodiazepin, cannabinoids, cocaine, morphine, phencyclidine and TBPE.

According to the present invention, as a label which can act as anindicator of presence or absence of an analyte in a liquid sample,fluorescent material is specifically used. The useful fluorescentmaterial may have a difference of 20 nm or more between its absorptionwavelength and emission wavelength. Representative examples of thefluorescent material include, but are not limited to, fluorescentparticles, quantum dots, lanthanide chelates, such as samarium (Sm),Europium (Eu) and Terbium (Tb), and fluors, such as FITC, Rhodaminegreen, thiadicarbocyanine, Cy2, Cy3, Cy5, Cy5.5, Alexa 488, Alexa 546,Alexa 594 and Alexa 647). Preferred fluorescent materials which can beused in detection of DNA are Cy3 and Cy5. In general, the fluorescenceintensity is directly proportional to the intensity of excitation light.

According to the present invention, the labeling material binds to thedetector which specifically binds to an analyte via a linker. Suchlinkers include, but are not limited to,N-[k-Maleimidoundecanoyloxy])-sulfosuccinimide ester (sulfo-KMUS),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy[6-Amidocaproate](LC-SMCC), K,K-maleimidoundecanoic acid (KMUA),succinimidyl-4-[p-maleimidophenyl]butyrate (SMBP),succinimidyl-6-[(β-maleimido-propionamido)hexanoate (SMPH),Succinimidyl-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB),sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-siab),N-[γ-maleimidobutyryloxy]-sulfo-succininimide ester (sulfo-GMBS),N-[γ-maleimidobutyryloxy]-succininimide ester (GMBS), succinimidyl3-(bromoacetamido) propionate (SBAP), N-β-maleimidopropionic acid(BMPA), N-[α-maleimidoacetoxy]succinimide ester (AMAS), N-succinimidylS-acetylthiopropionate (SATP), m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester(sulfo-MBS), N-e-maleimidocapric acid (EMCA),N-[e-maleimidocaproyloxy]succinimide ester (EMCS),N-succinimidyl-[4-vinylsulfonyl]benzoate (SVSB),N-[β-maleimidopropyloxy]succinimide ester (BMPS) and1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). Theselinkers will react with thiol groups of the detector.

The lateral flow assay strip of the present invention may take a shapeof a rectangle, circle, oval, triangle and other various shapes,provided that there should be at least one direction along which a testsolution moves by capillarity. In case of an oval or circular shape, inwhich the test solution is initially applied to the center thereof,there are different flow directions. However, what is taken intoconsideration is that the test solution should move in at least onedirection toward a predetermined position. Thickness of the stripaccording to the present invention is usually 0.1 to 2 mm, more usually0.15 to 1 mm, preferably 0.2 to 0.7 mm, though it is not important. Ingeneral, a minimum thickness is determined depending on a strength ofthe strip material and needs for producing a readily detectable signalwhile, a maximum thickness is determined depending on handling ease andcost of reagents. In order to maintain reagents and provide a sample ofa defined size, the strip is constructed to have a relatively narrowwidth, usually less than 20 mm, preferably less than 10 mm. In general,the width of the strip should be at least about 1.0 mm, typically in arange of about 2 mm to 12 mm, preferably in a range of about 4 mm to 8mm. The length of the strip is determined considering kinds of analytes,the number of test lines or spots and the number of reference lines onthe chromatography medium, space between pads, convenience of handlingand the like. Usually, it is 1 to 40 cm, preferably about 2 to 25 cm,more preferably about 4 to 20 cm. However, the strip can be practicallyprepared to have any length.

Solvents for a liquid sample to be analyzed are commonly aqueous media,which include oxidizing solvents having usually 1 to 6 carbon atoms,more usually 1 to 4 carbon atoms containing about 40 wt % or less ofanother polar solvent, particularly alcohol, ether, etc. In common, acosolvent is contained in an amount of less than about 20 wt %. Undersome circumstances according to the nature of an analyte, a part or allof the aqueous medium can be provided by the analyte per se.

The aqueous medium has pH of typically 4 to 11, more typically 5 to 10,preferably 6 to 9. The pH is selected in accordance with criticalbinding affinity sites of the binding elements and ability to maintainvoluntary generation of signals by a singal generation system. Variousbuffers can be used to adjust pH to a desired level and maintain pH atthat level during an assay. Representative buffers include for example,borate, phosphate, carbonate, Tris, and barbital. Though usable buffersare not particularly important, a certain buffer can be preferred forindividual assays as opposed to other buffers. Also, a non-ionicdetergent can be preferably added to the sample in an amount of about0.05 to 0.5 wt %. In addition, a variety of polyoxyalkylene compounds ofabout 200 to 20,000 Daltons can be used.

Typically, the assay is carried out at a mild temperature andpreferably, is carried out at a substantially constant temperature. Asuitable temperature for generating assay signal is usually about 4° C.to 50° C., more usually about 10° C. to 40° C., frequently ambienttemperature, i.e. about 15° C. to 25° C.

The concentration of the analyte to be analyzed in a subject solution istypically about 10⁻⁴ to about 10⁻¹⁵ M, more typically about 10⁻⁶ to10⁻¹⁴ M. The concentrations of other reagents is commonly determinedconsidering the concentration of a desired analyte and protocol.

In general, concentrations of various reagents in a sample and reagentsolution are determined in accordance with a concentration range of atarget analyte and a final concentration of each reagent is determinedempirically to optimize the sensitivity of the assay within a targetrange. Each reagent can be used in an excess amount along with a certainprotocol, as long as it does not lower the sensitivity of the assay.

Now, the package integratedly formed of the lateral flow assay strip andthe laser-induced epifluorescence detecting apparatus will be explainedin detail.

Backing of the Lateral Flow Assay Strip

The backing is typically made of water-insoluble, non-porous and rigidmaterial and has a length and width equal to the pads situated thereon,along which the sample develops, but may have a dimension being less orgreater than the pad. In preparation of the backing, various natural andsynthetic organic and inorganic materials can be used, provided that thebacking prepared from the material should not hinder capillary actionsof the absorption material, nor non-specifically bind to an analyte, norinterfere with the reaction of the analyte with a detector.Representative examples of polymers usable in the present inventioninclude, but are not limited to, polyethylene, polyester, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), nylon, poly(vinyl butyrate), glass, ceramic, metal andthe like.

On the backing, a variety of pads are adhered by means of adhesives.Proper selection of adhesives may improve the performance of the stripand lengthen the shelf life of the strip. According to the presentinvention, pressure-sensitive adhesives (PSA) may be representativelyused in the lateral flow assay strip. Typically, the adhesion ofdifferent pads of the lateral flow assay strip is accomplished as theadhesive penetrates into pores of the pads, thereby binding padstogether with the backing. With respect to such binding, ability of anadhesive to flow under normal conditions is referred to as “cold flow”.Since no heat is applied when coating PSA on to the pad, cold flow of acertain level is indispensable for binding between the pad and thebacking. If the level of cold flow is too low, the initial binding forceis low, causing insufficient binding between the pad and the backing. Incontrast, if the level of cold flow is too high, the adhesive migratesto the pads with which it is bound during storage of the strip, therebyclogging the pores, forming hydrophobic stains or leading to problems ofredamping the pad. Such problems associated with the cold flow of theadhesive can be solved by using a direct-casting membrane. For example,in the direct-casting membrane, a supporting plastic sheet prevents theadhesive from entering pores of the membrane and thus vertical migrationof the adhesive is prevented during storage.

Sample Pad of the Lateral Flow Assay Strip

The sample pad basically acts to receive the fluid sample containing ananalyte. Other than this function, the sample pad may have a function tofilter insoluble particles in the sample. From this point of view,preferred sample pads of the present invention are composed of cellulosefilter paper or glass fiber filter paper capable of providing thefiltering function. Usually, a cellulose membrane (grade 903) producedby S & S is used.

Preferably, the sample pad is treated in advance to prevent the analytein the sample from being non-specifically adsorbed thereto, to allow thecomponents of the sample to readily migrate through the chromatographymedium, to maintain the sensitivity of the reaction and to preventundesirable nonspecific reactions which may occur between the labeleddetector and components of the sample. The pretreatment of the samplepad is generally performed by treating the pad with an inactive proteinor surfactant. For instance, the pretreatment is carried out byimmersing the pad material in a solution of 0.1 to 10% bovine serumalbumin (BSA)-containing 0.1 M Tris buffer solution (pH 6-9), a solutionof 0.1% to 10% skim milk powder in 0.1 M Tris buffer solution (pH 6-9)and/or 0.1 to 10% casein solution. After leaving the sample pad as it isat 37° C. for 1 hour or at 4° C. for 1 day, the sample pad is removedfrom the solution and washed with a Tris buffer solution and dried. Thepretreatment with a surfactant is carried out by immersing the pad infor example, 0.01% to 1% solution of Triton X-100 or Tween 20, non-ionicsurfactant, followed by drying. Preferably, the sample pad may betreated with an inactive protein and then a surfactant. However, thesepretreatment steps are determined in accordance with kinds of analytesand samples.

Conjugate Releasing Pad of the Lateral Flow Assay Strip

On the conjugate releasing pad of the lateral flow assay strip accordingto the present invention, a fluorescently-labeled detector capable ofreacting with an analyte in the sample to form a conjugate is adheredbut is not immobilized. Since the detector is releasably attached, whenforming a conjugate via reaction with an analyte in the sample, it canmove together with the sample through the chromatography medium.

It is preferred for material of the conjugate releasing pad to have arapid filtering speed and a good ability to hold particles. As suchmaterial, synthetic material such as polyester and glass fiber filtercan be used. Commonly, glass fiber and polyester produced by S & S areused. Since these are biologically inactive and have more delicatefibrous material than natural material, they are not distorted orswollen when an aqueous reagent or sample is applied. Preferably, theconjugate releasing pad is pretreated with a reagent such as asurfactant so that an analyte is prevented from non-specifically bindingto the fluorescently-labeled detector on the releasing pad and theconjugate can smoothly be released and migrate.

Methods for attaching a reagent onto the conjugate releasing pad includean impregnation process in which a pad such as glass fiber is immersedin a solution of a high density reagent particularly formulated,followed by drying. However, the impregnation process has several simpleproblems. Firstly, the pad can be crumpled or distorted duringdehydration. Secondly, during drying the pad in a oven, reagents may beseparated from the pad or reconstituted due to surface tension andgravimetric action according to location on the pad. Thirdly, chemicalchanges of reagents may take place with the passage of time in theimmersion bath, causing the reagents to have different adsorption rates,whereby the reagents are unevenly coated on the pad. One method tominimize these problems is to perform drying of the pad in an oven atless than 40° C. for several hours. Another method is to lyophilize thepad instead of drying the pad in an oven. Such lyophilization ispreferred to drying in the oven in that stability of the detector can besecured.

As an alternative method to the impregnation process, a dispensingprocess may be used. This process involves dispensing 12 to 15 μl of areagent solution per cm of the pad using a dispenser and drying it. Thedrying of the pad is carried out the same as in the impregnationprocess. Also, the pad may be lyophilized.

Furthermore, the conjugate releasing pad may be treated with astabilizing agent and shielding agent. Examples of the stabilizing agentinclude saccharides such as sucrose, trehalose, etc. Examples of theshielding agent include proteins such as BSA (Bovine Serum Albumin),gelatin, casein, skim milk and the like, but are not limited thereto.

Chromatography Medium of the Lateral Flow Assay Strip

The material of the chromatography medium may be any one that can allowthe fluid sample and conjugate to rapidly move via capillary action toreach the captor immobilized thereon and preferably has homogeneousproperties. Typically, the chromatography medium refers to a porousmaterial having a pore diameter of at least 0.1μ, preferably 1.0μ andthrough which an aqueous medium can readily move via capillary action.Such material generally may be hydrophilic or hydrophobic, including forexample, inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly(vinyl chloride), polyacrylamide, cross-linked dextran, agarose,polyacrylate, etc.; either used by themselves or in conjunction withother materials. Also, ceramics may be used. The chromatography mediumcan be bound to the backing. Alternatively, the chromatography may bethe backing per se. The chromatography medium may be multifunctional orbe modified to be multifunctional to covalently bind to the captor.

When using a high concentration of the captor chemically binding to thechromatography medium so as to react and trap the analyte/detectorconjugate migrating from the conjugate releasing pad, preferably, anactivated filter paper sheet is used as the chromatography medium. Whena CNBr activated cellulose is selected as the material for the filterpaper, an activated cellulose filter paper sheet can be easily preparedby a known method such as the method described by Ceska and Lundkvist(Immunochemistry, 9, 1021 (1972)). When the material is DBM activatedcellulose, it can be easily prepared by a known method such as themethod described by Alwine (Methods Enzymol., 68, 220 (1979)). Further,a commercially available activated nylon film (Pall Immunodyne, USA) mayalso be used.

One of the important properties of the chromatography medium is itscapacity to immobilize a captor. Such binding capacity is varieddepending upon a pore structure of the medium and a post-treatment whichthe medium undergose. A preferred chromatography medium which can beused in the present invention is a nitrocellulose (NC) membrane andexamples thereof are described in Table 1 below. TABLE 1 Sec/4 cmSupplier Product (flow rate^((a))) IgG/cm^(2(b)) S&S AE 98 160-210 20-30ug (without a backing) AE 99 120-160 20-30 ug AE 100  90-120 20-30 ugMillipore HF 090  80-100 >95 (with a backing HF 120 107-133 >95 bound)HF 135 120-150 >95 HF 180 160-200 >95 HF 240 214-266 >120  Sartorius CN90 88-94 10-30 (with a backing CN 140 137-153 10-30 bound) CN 200206-233 10-30^((a))time for distilled water to move on the medium by 4 cm^((b))maximum binding capacity of IgG per cm²

In the table, the most preferred membrane is CN 90 membrane. Thismembrane has the smallest variation in flow rate among the describedproducts. The binding capacity on the order of 10 to 30 ug is sufficientsince amplification of fluorescent substances is excellent.

The captors are immobilized on the chromatography medium via chemicalbonding. The chemical bonding is carried out according to a known method(LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, Volume 15,Edited by R. H. BURDON and P. H. Van KNIPPENBERG ELSEVIER AMSTERDAM: NEWYORK, OXFORD (1985) P. 318-322). Further, the captor may be bound to theactivated paper sheet through a second substance (ex. antibody protein,etc.). When the second substance present therebetween is an antibody (tobe referred to as “second antibody” hereinafter), and for example, whenthe captor to be fixed is a monoclonal antibody derived from mouse,there may be used the activated paper sheet to which an excess ofanti-mouse γG (gamma globulin) hetero-animal antibody is bound and thena proper amount of the captor is bound by an immunoreaction. When thesubstance present therebetween is a protein, for example, there may beused the activated paper sheet to which an excess of protein A is bondedand then a proper amount of the trapping antibody is bound.

In order to uniformly wet test lines within the viewing window on thechromatography medium, a blocking technique is used. By blocking thechromatography medium with a material which can enhance rewetting of thechromatography medium, it is guaranteed that the medium can be rewetteduniformly and rapidly. There are four kinds of blocking material:proteins such as BSA and gelatin; surfactants such as SDS, Tween 20 andTriton X-100; polymers such as PVA, PEG and PVP. These blockingmaterials can be used at three points. Firstly, they can be applieddirectly onto the chromatography medium. This method can provide highlyuniform rewetting effects. However, the blocking should be performedafter immobilization of the captors but before adhesion of the samplepad. This method requires use of expensive coating equipments. Also, thecaptors should be redissolved and moreover, the blocking material maydeteriorate the antigenicities and storage life of the captors.Secondly, the blocking material may be incorporated into the sample pador conjugate releasing pad. This method has merits in that it can bereadily performed at low cost and re-dissolution of the captors is notneeded, but its blocking effect is not satisfactory. This method ispreferable in terms of easiness of handling though it has a blockingefficiency inferior to the first method. Thirdly, the blocking materialcan be added to a buffer solution to apply the captor on to thechromatography medium. This method also has merits in that it can bereadily performed at low cost and re-dissolution of the captors is notneeded. However, it has disadvantages that the captors tend to diffuseaway, the antigenicities and storage life of the captors may be lowereddue to the addition of the blocking agent.

In order to prevent non-specific binding of the reagents used in thesample pad, conjugate releasing pad and chromatography medium, two typesof methods may be used. One is to immerse the chromatography medium towhich the captors have been applied in a solution containing proteins ora highly polar polymer such as polyvinyl alcohol or to spray suchsolution to the chromatography medium to block up the non-specificbinding sites on the chromatography medium. However, this method maycause substitution of captors, particularly when the captors are notoptimally immobilized by complete drying before the blocking step. Theother method is to add a blocking agent to the sample pad. In this case,the blocking agent does not, at least initially, interfere with thebinding of captors to the chromatography medium. When the liquid sampleis applied to the sample pad, the blocking agent on the sample pad isresolubilized in the sample pad, to thereafter move together with thesample. Ideally, a sufficient amount of a blocking agent is added to thesample pad to block all non-specific bindings of all the analytes anddetectors.

According to the preferred embodiment of the present invention,biotin-avidin conjugate is used as an analyte capture system. That is,avidin is added onto and immobilized on the test line of the stripinstead of a captor (ex. antibody or antigen protein). Biotin isattached to the captor. As the biotin binds to the avidin, the captorcan be automatically detected on the test line of the strip. When thecaptor is a protein, the binding of the biotin to the captor is effectedby reacting with an amine group of lysine or arginine among amino acids.Thus, the biotin is specifically bonded to a certain site of the proteinvia an amine group of such amino acids. The protein with biotin attachedspecifically binds to the avidin which has been planted and immobilizedon the strip and always maintains one orientation. Further, since thecaptor is not fixed via adsorption, the protein of the captor is notchanged in structure or functions thereof. For these reasons, unlike theconventional method by which the captor is immobilized non-specificallyon the layer of the strip without maintaining a particular orientation,according to the biotin-avidin method, the captor protein can beimmobilized in one direction, and thereafter always maintain theorientation. Also, since the proteins of the captors are not changed intheir structure or functions by the adsorption, they can moreeffectively react with the analytes in the test sample. Therefore, thebiotin-avidin method can exhibit much higher sensitivity at the sameconcentration of an analyte compared to the conventional method. Evenwhen an analyte is present at a concentration 10 to 100 times lower thanthe immobilized captor in the conventional method, a high sensitivitycan be attained. Further, with an avidin-immobilized strip, when adifferent analyte is examined, there is no need for preparing a newstrip of a different type. Only different protein-biotin conjugates andprotein-fluorescently labeled conjugates as detectors are needed toassay various analytes. The method according to the present inventioncan be performed using a strip having the conjugate pad as in theconventional method, but also can be performed by directly addinganalytes, protein-biotin conjugates, and protein-fluorescent conjugatesin a solution to the sample pad without a conjugate pad. FIG. 17 showsthe schematic diagram of the method using the biotin-avidin system onthe test line of the strip and the conventional method.

Absorption Pad of the Lateral Flow Assay Strip

The absorption pad is means for physically absorbing the sample whichhas chromatographically moved through the chromatography medium viacapillary action and for removing unreacted substances. Thus, theabsorption pad is located at the end of the lateral flow assay strip tocontrol and promote movement of samples and reagents and acts as a pumpand container for accommodating them. The speeds of samples and reagentsmay vary depending on the quality and size of the absorption pad.Commonly used absorption pads are formed of water-absorbing materialsuch as cellulose filter paper, non-woven fabric, cloth, or celluloseacetate.

Wicking Pad of the Lateral Flow Assay Strip

The wicking pad is used to improve diffusion of a sample such as bloodand prevent contamination of the conjugate pad by microorganisms duringstorage. Commonly, AW14-20T4, hydroxylated polyester, produced by PallCorporation (USA) is used.

Now, the lateral flow assay strip according to the present inventionwill be more concretely described while referring to the appendedfigures. From this description, the features and advantages of thepresent invention will become more apparent.

FIG. 1 and FIG. 3 show the conventional lateral flow assay strip.Referring to FIG. 1, the lateral flow assay strip 1 includes a samplepad 2 attached to one end of a backing 6 via an adhesion layer, uponwhich a liquid sample containing an analyte is applied, andsuccessively, a conjugate releasing pad 3, a chromatography medium 4 andan absorption pad 5 toward the opposite end of the sample pad. On theconjugate releasing pad 3, a labeled detector is releasably attached sothat the analyte in the liquid sample chromatographically moving viacapillary action can react with the detector to form conjugate. On thechromatography medium 4, a captor which is identical to or differentfrom the detector is immobilized in a line (test line) 11 by chemicalbonding. The captor in the line 11 chemically reacts with the liquidsample and the conjugate formed on the conjugate releasing pad 3, whichhave been chromatographically moving on the strip, trapping theconjugate to form a labeled detector/analyte/captor conjugate. Remainingunreacted substances and the liquid sample continuously move bycapillary action on the strip and are absorbed by the absorption pad 5.The amount of the analyte is determined by measuring the amount of theconjugate. The amount of the conjugate is determined as a relative valueby comparing a luminescent intensity of the conjugate trapped on thechromatography medium with a reference luminescent intensity which isobtained from a conjugate formed of a reference detector which has beenlabeled identically with the analyte detector, and is different from theanalyte detector and captor, and a reference captor which is not labeledand is different from the reference detector.

As described above, the conventional lateral flow assay strip isdesigned with the intention of quantitatively analyzing only one kind ofan analyte in a biological sample.

FIG. 2, FIG. 4 and FIG. 5 show a lateral flow quantitative assay stripof an embodiment according to the present invention. The lateral flowassay strip has a construction in which four or more kinds of detectorsare releasably attached on the conjugate releasing pad 2, and variouscaptors in the same number as the kinds of the detectors are immobilizedin micro test lines 11, whereby it is possible to assay diverse analytesat the same time. Here, each kind of captor is immobilized in anindividual test line and hence, the number of the test lines are thesame as the number of the kinds of captors. That is, it is possible toassay as many analytes as the number of the test lines. The number ofthe test lines can be restricted by the size of the strip, kinds andamounts of reagents (detectors and captors) used in the strip, requiredsensitivity, etc. According to the present invention, the strip hasgenerally 2 to 20 test lines, preferably 5 to 15 test lines.

FIG. 6 is a lateral flow quantitative assay strip of another embodimentaccording to the present invention. The lateral flow quantitative assaystrip as shown in FIG. 6 includes a sample pad 2 disposed on achromatography medium 4, a conjugate releasing pad 3 disposed on thesample pad 2 and a wicking pad 8 disposed on the conjugate releasing pad3. The wicking pad 8 mounted on the conjugate releasing pad 3 is tofacilitate dispersion of the liquid sample, to enhance the solubility ofthe conjugate releasing pad and to protect detectors and referencedetectors which are releasably attached to the conjugate releasing padfrom being modified or damaged by microbial contamination during storageof the strip.

The representative wicking pads which are useful according to thepresent invention are ones formed of hydroxylated polyesters, but arenot limited thereto. Among examples of such hydroxylated polyesters isAW14-20T4, produced by Pall Corporation (USA).

After application to the wicking pad 8, the liquid sample is dispersedwhile passing through the wicking pad 8 and diffused to the conjugatereleasing pad 3, upon which detectors and reference detectors releasablyattached to the conjugate releasing pad 3 are dissolved in the liquidsample and react with analytes in the sample, forming conjugates. Theconjugates along with the liquid sample then reach the sample pad 2. Thesample pad 2 disposed under the conjugate releasing pad 3 acts assupplementary means, in which the analytes in the liquid sample whichhave not yet formed conjugates can sufficiently reacts with unreactedreagents. Also, the sample pad 2 acts as buffering means so that theliquid sample and conjugates can smoothly moves to the chromatographymedium 4. Next, the formed conjugates moves to the chromatography mediumalong with the liquid sample which moves to the chromatography mediumvia capillary action, to be trapped by reagents (captors and a referencecaptor) which are immobilized in test lines on the chromatographymedium.

FIG. 7 is a lateral flow quantitative assay strip of yet anotherembodiment according to the present invention. This strip is onlyprovided with a sample pad 2 on a chromatography medium 4. Here, thesample pad 2 also can serve as a conjugate releasing pad sincefluorescently-labeled detectors and a reference detector are releasablyattached to it. The formed conjugates formed on the sample pad move tothe chromatography medium along with the liquid sample which movesthrough the chromatography medium via capillary action, to be trapped byreagents (captors and a reference captor) which are immobilized in testlines on the chromatography medium.

In another mode, the sample pad does not have detectors and a referencedetector attached to it. In this case, a liquid sample is applied on thesample pad in a two-step process, by which the liquid sample is mixedwith fluorescently-labeled detector and a reference detector and themixture is applied to the sample pad. This two-step process provideseffects of increasing the sensitivity of the assay by a factor of two ormore compared to the method in which the fluorescently-labeled detectorand a reference detector are attached to the sample pad. However, it hasa disadvantage in that the detectors should be stored in a refrigeratorbefore use, causing problems in portability. Analytes in the liquidsample react with the detectors to form conjugates. The formedconjugates moves to the chromatography medium along with the liquidsample which moves to the chromatography medium via capillaryphenomenon, to be trapped by reagents (captors and a reference captor)which are immobilized in test lines on the chromatography medium.

FIG. 8 is a lateral flow assay strip of yet another embodiment accordingto the present invention. This lateral flow assay strip includes aconjugate releasing pad 3 disposed on a chromatography medium and asample pad 2 disposed at a fixed position on the conjugate releasing pad3. After application to the sample pad 2, the liquid sample migrates tothe conjugate releasing pad 3 by diffusion, upon which analytes ifpresent in the liquid sample react with fluorescently-labeled detectors,forming conjugates. The formed conjugates moves to the chromatographymedium along with the liquid sample which moves to the chromatographymedium via capillary action, to be trapped by reagents (captors and areference captor) which are immobilized in test lines on thechromatography medium.

FIG. 9 is a lateral flow assay strip of yet another embodiment accordingto the present invention. This lateral flow assay strip is the same asthat shown in FIG. 6, except that it has two layers of chromatographymediums laminated together. A second chromatography medium 13 is adheredto an adhesive layer 7 on a backing 6 and a first chromatography medium4 is disposed on the second chromatography medium 13, extending overboth ends of the second chromatography medium 13, in which the extendedends of the first chromatography medium 4 are adhered to the adhesivelayer 7 on the backing 6. On the first chromatography medium 4, a samplepad 2 and an absorption pad 5 are disposed at its opposite ends,respectively.

Captors 11 and reference captors 9,10 are immobilized at the upper andlower part, respectively, of the interface between the firstchromatography medium 4 and the second chromatography medium 13. Here,the captors immobilized at the bottom of the first chromatography mediummay be identical to or different from the reference captors immobilizedat the top of the second chromatography medium. If identical referencecaptors are immobilized on the interface between the two chromatographymedia, the sensitivity is increased by at least two times, compared towhen the reference captors are immobilized on one chromatography medium.If different reference captors are immobilized on the interface betweenthe two chromatography media, it is possible to assay twice the numberof analytes compared to when identical reference captors are immobilizedon the interface between the two chromatography media, and when thereference captors are immobilized on one chromatography medium.

Also, this type of lateral flow assay strip may be advantageous in thatit can be stored for an lengthy period of time while maintaining itssensitivity, since the reagents are not exposed to the outside.

FIG. 10 is a lateral flow assay strip of yet another embodimentaccording to the present invention. This lateral flow assay stripincludes a thin and transparent polyester film on the lateral flow assaystrip shown in FIG. 6. By laminating a polyester film on the top of thestrip, it is possible to reduce the background signal by labeling due towater evaporation occurring during the test and thus to maintain a highsensitivity. Also, the top of the strip which is exposed to theatmosphere during testing or storage can be protected from moisture andairborne contaminants by the laminated polyester film, thereby providinga consistent quality. Further, an acryl adhesive tape can be adhered tothe top of the strip, which can solidly integrate the pads adhered tothe backing of the strip in addition to the foregoing effects obtainedby the polyester film.

FIG. 11 is a lateral flow assay strip of yet another embodimentaccording to the present invention. This lateral flow assay stripfurther includes a thin and transparent polyester film or an acryladhesive tape on the top of the lateral flow assay strip shown in FIG.9. This type of strip provides the advantages of the lateral flow assaystrips shown in FIG. 9 and FIG. 10, thus being very preferred.

Laser-Induced Epifluorescence Detecting Apparatus

The present invention, in general, relates to a method for detectinglaser-induced epifluorescence and a device therefor. More particularly,the present invention relates to a method for detecting epifluorescenceby using an elliptical or spherical reflecting mirror which caneffectively collect fluorescence emitted when irradiating laser or lightwith a proper wavelength to a fluorescent substance attached on thesurface of an opaque sample transparent and a device therefor.

A representative method used for detecting fluorescence of DNA chips orprotein chips using membranes is laser-induced fluorescing detection.The laser-induced fluorescing detection involves rendering a fluorescentmaterial to be an excited state using a laser as a excitation lightsource and measuring the intensity of fluorescence emitted when thematerial returns to the ground state, in which the fluorescenceintensity can be converted to a concentration of the material. Thus, itis possible to quantify DNA or protein sample by attaching a fluorescentmaterial.

One of the most commonly used detectors which apply the laser-inducedfluorescence detection is a confocal laser scanning system. In thissystem, a laser is used as a light source and fluorescent signalsemitted from a specimen are focused on to a photomultiplier tube as aseparate special detector, in which the signals are converted intodigital images. Thus, the confocal laser scanning system irradiates alight of only the wavelength range suitable for the fluorescent materiallabeling the specimen using a laser to induce emission of fluorescence.In this case, various kinds of filters such as a beam splitter can beselected and a pinhole is located at the last position in front of thedetector so that the detector receives only the image being in completefocus (FIG. 1). Meanwhile, when using the confocal laser scanningsystem, it requires to use a cover glass of a proper thickness and toselect a suitable object lens and mounting medium. It has an advantagethat images being out of focus can be removed.

In laser-induced fluorescence detection devices and technologies at thepresent time, it is important to improve the sensitivity of thefluorescence detectors. One of the main factors in improving thesensitivity of the fluorescence detectors is to collect maximumfluorescing radiation, while minimizing the background, i.e., collectedexcitation light intensity).

Therefore, in order to obtain optimum detection limit in a laser-inducedfluorescence detector, the fluorescence generated from afluorescently-labeled sample must be collected with high efficiency,while scattered excitation light reaching the detector must beminimized. In practice, high aperture value objectives or reflectors maybe used to collect fluorescence. The fraction of light collected by alens is related to the aperture value of the lens, and refractive indexof the surrounding medium. This relationship may be expressed by thefollowing equation:Collection Efficiency=Sin²(Sin⁻¹ NA)=Sin²(Sin⁻¹(½F))

In the above equation, F represents the aperture value (F number) and NArepresents numerical aperture. Collection efficiency of 1 implies thatthe lens collects all of the light emitted by the sample.

Usually, the light collecting lens is surrounded by air, which has arefractive index of 1. From the above equation, it can be seen that alens of a very small aperture value is required to obtain highcollection efficiency. A lens with an aperture value of 1 will collect50% of the light emitted by the sample. Although lenses immersed in aliquid medium (oil, water, etc.) can have an aperture value less than 1,they collect less than 50% of the emitted light because the refractiveindex of the immersion fluid is usually larger than the refractive indexof the lens material. In addition, according to the above equation, alens with an aperture value of 0.5 and located in air collects only 7%of the emitted light. In deed, it is impossible to have an aperturevalue of 0.5.

In the conventional arts, refractive and/or reflective opticalcollectors are utilized in laser-induced fluorescence detectors with orwithout optical fibers for collecting the emitted fluorescing radiation.However, the capability of these collectors collecting fluorescencelight is limited by their maximum collection angle. A typical highcollector will have a collection cone angle of about 90°. Thiscorresponds to a 0.7 numerical aperture, or 14% collection efficiency.

U.S. Pat. No. 5,614,726 issued to Kaye et al. on Mar. 5, 1997 discloseda laser-induced fluorescence detector. However, the detector has acollection efficiency of only 6 to 7%.

Therefore, there exists a need to develop a improved laser-inducedfluorescence detector that has a higher collection efficiency optics andenhanced signal strength in detection of epifluorescence of lateral flowassay strips and biochips such as DNA chips or protein chips usingmembranes.

According to the present invention, in order to measure epifluorescenceto determine quantities of analytes, a laser-induced epifluorescencedetecting apparatus is used, which comprises a laser, an exciter filter,an elliptical reflecting mirror or spherical mirror, epifluorescencesample control means, a collimator, a fluorescent filter and an opticaldetector, in which the components are arranged in a structure such thatlight emitted from a laser is passed through the exciter filter andfocused to the surface of a sample, in which the focusing point is thefirst focal point of the elliptical reflecting mirror with a propersize, scattered light of the incident light and fluorescence emittedfrom the first focal point are reflected from the elliptical reflectingmirror and focused upon a spatial filter as the second focal point ofthe elliptical reflecting mirror, and the light from the spatial filteris converted into parallel light by a collimator and passed through afluorescent filter to remove the scattered light, thereby providing apure fluorescence component to the optical detector.

A conventional lateral flow assay strip comprises a sample pad, to whicha sample is applied, a releasing pad coated with a detector antibody, adeveloping strip (typically, nitrocellulose), in which components of thesample move at different rates to be individually separated and toundergo antibody-antigen reaction and an absorption pad provided at thefar end of the sample pad to cause the sample to keep moving.Particularly, the lateral flow assay strip has a sample pad 2 attachedto one end of a support via an adhesion layer, upon which a liquidsample containing an analyte is applied, followed by a conjugatereleasing pad 3, a chromatography medium 4 and an absorption pad 5toward the opposite end of the sample pad. On the conjugate releasingpad, a first fluorescently-labeled detector is releasably attached sothat the analyte in the liquid sample, while chromatographically movingvia capillary action, can react with the detector to form conjugate. Onthe chromatography medium, a second captor which is identical to ordifferent from the first detector is immobilized in a single line (testline) by chemical bonding. The captor in the single line chemicallyreacts with the liquid sample and the conjugate formed on the conjugatereleasing pad, which have been chromatographically moving on the strip,trapping the conjugate. Remaining unreacted substances and the liquidsample continuously move by capillary action on the strip and areabsorbed by the absorption pad. The amount of the analyte is determinedby measuring the amount of the conjugate, which is determined as arelative value by comparing a luminescent intensity of the conjugatetrapped on the chromatography medium with a reference luminescentintensity which is obtained from a conjugate formed of a third detectorwhich is different from the first detector and the second captor and hasbeen labeled identically with the first detector and a unlabeled forthcaptor which is different from the third detector.

DNA chip refers to an array of hundreds to hundreds of thousands of DNAspacked in a very small space by mechanical automation and electroniccontrol. In other words, the DNA chip is a biological chip constructedby binding DNAs to a small substrate of glass, silicone, or nylon toanalyze gene expression aspects, gene binding, protein distribution,reaction aspect, etc. The DNA chips are classified into cDNA chips andoligonucleotide chips according to the size of genetic substance bindingto a substrate. The cDNA chips have genes (full-length open leadingframe) of at least 500 bp while the oligonucleotide chips haveoligonucleotides of about 15 to 25 bp.

Techniques for manufacturing DNA chips using target DNA are largelydivided into two types: one is to directly synthesize oligonucleotideson a substrate and the other is to plant synthesized or amplified targetDNA onto a substrate. The former applies the photolithographic methodderived from manufacturing method of semiconductor chips and has anadvantage that it can accomplish high-density integration. However, thesize of the target DNA is limited to 20 nucleotides. It is suitable fordisease diagnosis or research of single nucleotide polymorphism (SNP).The latter DNA chip is applied in researches of differential geneexpression. The target DNA is planted on a slide coated with polyL-lysine, amine or aldehyde.

Methods for plotting DNA include the micropipetting using apiezoelectric method. Spots have a diameter of about 100 μm anddistributed at a ratio of about 1000 spots/cm².

Methods for labeling nucleic acid in a DNA chip are largely divided intodirect labeling and indirect labeling though there are diverse methods.The direct labeling involves direct insertion of fluorescent tags into aprobe mixture of nucleic acid to be hybridized in a DNA chip by enzymesynthesis. The direct labeling has merits that it is simple, providesstrong hybridization signal and can use other dyes having similarchemical structures but different in their absorption or emissionwavelengths. Such merits are important, particularly in comparativeanalyses, because a fluorescent material having a much differentstructure may cause an erroneous result in a differential geneexpression experiment. In the direct labeling, it is necessary to usedifferent kinds of fluorescent materials for maximization of precisionin a comparative analysis. For DNA chips, chemically related dyes suchas cyanine and alexa analogs are used. The indirect labeling isaccomplished by inserting an epitope into a probe mixture of nucleicacid. After hybridization of tagged nucleic acid with the epitope, theDNA chip stains proteins bound to the epitope. By virtue of the stainedprotein, fluorescent signal can be obtained. One of general examples ofthe indirect labeling is to use the binding of biotin epitope andfluorescent streptavidin-phycoerythrin. The greatest advantage of theindirect labeling is that signal amplification increases 10 to 100 timeshigher, compared to the direct labeling. Capital shortcomings of theindirect labeling are that it is difficult to be performed, the epitopehas different labeling efficiencies and the precision is poor incomparative analyses due to the binding force of proteins.

A format of the most commonly used DNA chips is a 1″×3″ microscopeslide. Since it has a large surface area (approximately 19 cm²), it canproduce 100,000 or more arrays using microspotting and ink-jettingtechniques. Also, due to the peculiarly low fluorescence intensity ofglass per se, it can be favorably used. In addition, a cassette,produced by Affymertix is widely used as a DNA chip format. The cassetteof affymertix is enclosed in a plastic frame to protect a glass chip.

According to the present invention, it is possible to produce veryabundant data from a DNA chip, which can serve as a basis ofbioinformatics. Data can be applied to a modeling, yielding muchinformation via computational biology. Quantified data from DNA chipsare displayed by various methods, among which a representative method isa scatter plot. Plots of the whole data obtained from a two-colorexperiment are expressed as a function of ratio and signal intensity.When the ratio is greater than 1.0, the result is plotted above thediagonal where the ratio is 1.0 and when the ratio is less than 1.0, theresult is plotted below the diagonal. This method can be used as meansto survey a huge database such as thousands of gene expression profiles.Commonly, with one mouse click of a spot on a computer screen,information of the gene sequence corresponding to the spot in the DNAchip is displayed.

In another aspect of the present invention, there is provided a methodfor detecting epifluorescence emitted from a protein chip. Preparationsand applications of the protein chip are well-known to those skilled inthe art and diversely described in numerous science journals andpatents. For example, the protein chip comprises antibodies againstproteins associated with various diseases which are integrated on asmall substrate. It can diagnose early presence and progress of diseaseusing analytes prepared from body fluid of a patient as a marker. As thesubstrate, common glass plates may be used and for example, avidin isused to attach desired proteins to a glass plate. Alternatively,polystyrene may be used as substrate material. The polystyrene substratehas an advantage that proteins can be readily attached with highattachment efficiency. In addition, a polyvinyl chloride orpolypropylene substrate can be used according to natures of proteins tobe attached to the substrate.

Processes for integrating proteins on the above-described substrates arewell known to those skilled in the art. For example, when using apolystyrene substrate, 8 grooves a width of 1 mm, a length of 2 mm and adepth of 1.5 mm are formed at intervals of 1 mm on a 1.5 cm×1.5 cmpolystyrene substrate. Proteins to be analyzed are deposited asagglomerates spaced from each other with a predetermined distance ineach groove. For instance, where proteins are deposited as agglomerateswith a diameter of 400 nm at intervals of 500 nm, 10 different proteinscan be deposited over a length of 1 cm. That is, 80 different proteinscan be deposited in a substrate.

As fluorescent material which can label an analyte in a liquid sample inthe laser-induced epifluorescence detecting method and apparatusaccording to the present invention, fluorescent materials having adifference of 20 nm or more between its absorption wavelength andemission wavelength may be used. Representative examples of suchfluorescent materials include, but are not limited to, fluorescentparticles, quantum dots, lanthanide chelates, such as samarium (Sm),Europium (Eu) and Terbium (Tb), and fluors, such as FITC, Rhodaminegreen, thiadicarbocyanine, Cy2, Cy3, Cy5, Cy5.5, Alexa 488, Alexa 546,Alexa 594 and Alexa 647). Preferred fluorescent materials which can beused in detection of DNA are Cy3 and Cy5. In general, the fluorescenceintensity is directly proportional to the intensity of excitation light.

The lasers which can be used in the laser-induced epifluorescencedetecting method and apparatus according to the present inventioninclude He—Ne lasers and diode lasers. Examples of the He—Ne lasers mayinclude an accurate and downsized portable iodine-stabilized He—Ne laserdeveloped in cooperation with the National Research Laboratory ofMetrology (NRLM), Agency of Industrial Science and Technology (AIST),ministry of International Trade and Industry (MITI) and model 05 LYR173, produced by Melles Griot (Irvine, Calif.). The diode-laser is moreaccurate and compact than the He—Ne laser and includes infrared and reddiode lasers.

The laser-induced epifluorescence detecting apparatus used in thepresent invention comprises a laser, an exciter filter, an ellipticalreflecting mirror or spherical mirror, epifluorescence sample controlmeans, a collimator, a fluorescent filter and an optical detector, asdescribed above. Now, the principle of collecting epifluorescence bysuch construction will be explained referring to FIG. 12 and FIG. 13while comparing with the conventional epifluorescence detectingapparatus.

FIG. 12 shows a laser-induced epifluorescence detecting apparatus withan elliptical reflecting mirror, in which incident light is focused uponthe first point of the elliptical reflecting mirror where the sample isdisposed and the light focused upon the second point of the ellipticalreflecting mirror is converted into parallel light to detect theepifluorescence. The laser can emit light at a wavelength correspondingto the maximum excitation band of a certain fluorophore. In oneembodiment, the laser 21 is a 2 mW He—Ne laser emitting light at awavelength of 594 nm which is approximate to the maximum absorption ofAlexa, a fluorescence-labeling substance. For example, a He—Ne lasermodel 05 LYR 173 (Melles Griot, Irvine Calif.) may be used. Light fromthe laser 21 passes through the incident filter (exciter filter) 22 andthen a hole disposed at the middle point between the incident filter 22and an elliptical reflecting mirror 23. Here, the elliptical reflectingmirror 23 is preferably aligned in a straight line with the incidentfilter 22 so that the light from the laser can properly pass through theincident filter 22 and then the elliptical reflecting mirror. Afterpassing through the elliptical reflecting mirror, the light is focusedto the surface of the sample (DNA chip or protein chip) fixed on thesample control means 24. The sample may be moved upward and downward orback and forth to a proper position so that the light can be optimallyfocused to the surface of the sample. The focal point is located at thefirst point of the elliptical reflecting mirror 23. The fluorescenceemitted from the sample positioned at the first focal point of theelliptical reflecting mirror and scattered light of the incident lightare reflected by the elliptical reflecting mirror 23 and focused to thespatial filter 25 at its second focal point. The spatial filter servesto remove noise created by dust, etc. on the surface of the sample.After passing the spatial filter, the light is converted into parallellight by the collimator 26. The parallel light is filtered again throughthe fluorescent filter 28 to remove the scattered light andsubsequently, pure fluorescence components enter the optical detector27. In the optical detector 27, the fluorescence components areconverted into signals expressing their fluorescence intensities, whichare transmitted to a computer 29, in which they are processed.

FIG. 13 shows a laser-induced epifluorescence detecting apparatusaccording to the present invention with an spherical reflecting mirror,instead of the elliptical reflecting mirror as shown in FIG. 2, in whichfluorescence of the sample obtained by focusing incident light to thesurface of the sample is focused to the image point of the sphericalmirror and the light is converted into parallel light for detection ofthe epifluorescence. The procedure for detecting epifluorescence of thesample using the apparatus of FIG. 13 is the same as described for theapparatus of FIG. 12. Light from the laser 31 passes through theincident filter (exciter filter) 32 and then a hole disposed at themiddle point between the incident filter 32 and a sample control means34. Here, the spherical reflecting mirror 33 is preferably aligned in astraight line with the incident filter 32 so that the light from thelaser can properly pass through the incident filter 32. After passingthrough the spherical mirror, the light is focused to the surface of thesample fixed on the sample control means 34. The sample may be movedupward and downward or back and forth to a proper position so that thelight can be optimally focused to the surface of the sample. The focalpoint is located at the first point of the spherical mirror 23. Thefluorescence emitted from the spherical mirror and scattered light ofthe incident light are reflected by the elliptical mirror 33 and focusedto the spatial filter 35 at its second focal point. The spatial filterserves to remove noise created by dusts, etc. on the surface of thesample. After passing the spatial filter, the light is converted intoparallel light by the collimator 36. The parallel light is filteredagain through the fluorescent filter 38 to remove the scattered lightand subsequently, pure fluorescence components enter the opticaldetector 37. In the optical detector 37, the fluorescence components areconverted into signals expressing their fluorescence intensities, whichare transmitted to a computer 39, in which they are processed.

Meanwhile, according to the conventional epifluorescence detectingapparatus as shown in FIG. 21, light from a laser passes through anaperture 130 and then is reflected by a beam splitter 160. The reflectedlight passes through a lens 150 and then is focused to the surface ofthe sample. The fluorescence emitted from the surface of the sample 140passes again through the lens 150 and to be focused to an aperture 120before entering a detecting apparatus 170. In this case, since the areain front of the sample 140 is exposed to the outside, a great quantityof the fluorescence emitted from the surface can be scattered to the airto be lost, not passing through the lens. As contrast, in the detectingapparatuses according to the present invention, epifluorescent samples24, 34 are located in the concave region of the elliptical reflectingmirror 23 or spherical mirror 33. Therefore, the great majority offluorescence from the samples can be reflected by the ellipticalreflecting mirror 23 or spherical mirror 33, not being scattered to theair and thereby, there is little chance for fluorescence to be lost.Moreover, in the epifluorescence detecting apparatus according to thepresent invention, since the collimator 26, 36 is used to convert thefluorescence reflected by the elliptical reflecting mirror or sphericalmirror 33 into parallel light, there is little chance for signal to belost on the way to the optical detector 27, 37.

In sum, the laser-induced epifluorescence detecting apparatus accordingto the present invention employs a reflective surface which surroundsthe sample, instead of a lens, to focus the epifluorescence, andconsequently, 97% or more of the emitted epifluorescence can bereflected by the reflective surface toward the optical detector(excluding the loss due to the hole through which the incident lightenters). The sample and the sample control means shield a part of thereflected epifluorescence, but the remaining epifluorescence with theexception of such inevitable losses due to the structure of theapparatus all reaches the optical system. When the diameter of thereflecting mirror is 10 cm and the size of the sample control means is2×10 cm, the loss due to blocking by the sample control means is on theorder of about 20%, which includes the loss by the hole for entry ofincident light. Therefore, according to the present invention, it can beexpected that the fluorescence collecting efficiency is raised to 80% ormore over 10% of the conventional lens type fluorescence collectingdevice.

FIG. 23 is a view illustrating a structure of a sample control means inan embodiment of the present invention. In FIG. 23, the holder 41consists of the upper part into which one end of a sample (mounted onstrip or chip) is inserted and fixed and the lower part connects with apitch wheel of a moving means 42 so that the sample can move in frontand rear directions of the pitch wheel. As the moving means 42 spins bya motor 45, it can move in front and rear directions to keep up with aguide 43 equipped side by side by the moving means 42.

In general, cut-offs of analytes to be analyzed in blood are describedin Table 2 (microcystin is an environmental material, existing in waterbut not in blood). TABLE 2 Marker Unit Cut-off CEA ng/ml <5 AFP ng/ml<15 PSA ng/ml <4 B2M ng/ml <2 NSE ng/ml <15 CYFRA21-1 ng/ml <3.5Myoglobin ng/ml <70 CK-MB ng/ml <3 Ctnl ng/ml <1 CTnT pg/ml <60 BNPpg/ml <100 Microcystin pg/ml <300

In an additional embodiment according to the present invention, analyteswhich can be analyzed at a level of pg/ml include those described inTable 3, but are not limited thereto. Analytes Unit Cut-off ACTH pg/ml200-250 Adrenomedullin pg/ml  480 ± 135 ANP pg/ml 73 Angiotensin IIpg/ml 21 ± 4 Calcitonin pg/ml 10 CNP pg/ml 7.36 ± 3.0 Endorphin pg/ml 30± 5 Gastrin pg/ml 26.4 ± 8.4 Ghrelin pg/ml  87.79 ± 10.27 NPY pg/ml 70.7± 5.9 Pancreatic polypeptide pg/ml 218 ± 23 Urotensin pg/ml  7.70 ± 0.97

It has been found that the laser-induced epifluorescence detectingapparatus according to the present invention can assay analytes to apg/ml level.

Now, the present invention will be described in detail using anembodiment shown in the following examples. However, the examples arefor illustration of the present invention and do not limit the scope ofthe present invention thereto.

EXAMPLE 1 Preparation of Monoclonal Antibody for Use as Detector andCaptor

(1) Preparation of Culture Medium

Powdered Dulbecco's modified Eagle's media (DMEM) was dissolved in 900ml of DDW and 3.7 g of sodium bicarbonate was added to the solution toadjust pH to 6.9. The solution was sterilized using a filter having apore size of 0.45 μm, thus obtaining “incomplete DMEM”. 450 ml of theincomplete DMEM was supplemented with 10% bovine calf serum andantibiotic penicillin-streptomycin to obtain “complete DMEM”. 5 ml ofthe complete DMEM was mixed with 5 ml of 100×HT to form HT(hypoxanthine+thymidine) and 5 ml of 100×HAT(hypoxanthine+aminopterin+thymidine) to form a HT medium and HAT medium,respectively.

(2) Preparation and Injection of Antigen

For the first injection, purified enzyme protein solution (50 μg) wasmixed with an equal volume (typically 0.3 ml) of complete Freund'sadjuvant and the mixture was subjected to sonication for 30 seconds. Theresulting solution was injected to BALB/c mice at a dose of 0.4 ml.Three weeks after the first injection, the additional injection wasperformed to the mice using a solution prepared by mixing the proteinsolution used for the first injection with incomplete Freund's adjuvant.This booster injection was repeated 2 or 3 times. The final injectionwas performed using only the protein without any adjuvant 3 to 4 daysbefore a cell fusion experiment. The mice used in the experiment were 6to 8 weeks old BALB/c, without distinction of sex.

(3) Preparation of Feeder Cells

The feeder cells were prepared 1 to 2 days before the fusion experiment.A mouse, at least 10 weeks old, was sacrificed and the abdominal skinwas removed with great care. 5 ml of 11.6% sugar solution was injectedintraperitoneally. 1 or 2 minutes later, the injected sugar solution wasrecovered in an amount of at least 3 ml. The solution was centrifuged(2,000 rpm, 3 minutes) to obtain feeder cells. The feeder cells weresuspended in 30 ml of HAT medium and the resulting solution was placedin five 96-well plates, one drop for each well. When the mouse wassmall, two mice were used to obtain abdominal cells. Also, whencontaminating red blood cells were present, the preparation wasrepeated.

(4) Preparation of Spleen Cells

A mouse immunized with an antigen was sacrificed and its spleen wasremoved under sterile conditions. The spleen was transferred to aculture dish to which 10 ml of incomplete DMEM had been added in advanceand its tissue was disrupted with tweezers, upon which the spleen cellswere released to the culture medium. The cells were moved to a 15 mltube to settle any large or uncrushed tissues for 2 minutes. A 5 mlaliquot from the upper part was centrifuged. The supernatant was removedand the cells were dissolved into 3 ml of incomplete DMEM, which was tobe mixed with myeloma cells. For one cell fusion experiment, 3×10⁷spleen cells were prepared.

(5) Preparation of Myeloma Cells

At 5 days before the cell fusion experiment, SP2/0 Ag14 cells in frozenstate were taken out of a liquid nitrogen tank and thawed. The cellswere recovered while very slowly adding complete DMEM. Centrifugationwas performed to settle the cells, which were resuspended in 10 ml ofcomplete DMEM and passaged at intervals of two days in a CO₂ incubatorat 37° C. 5×10⁷ myeloma cells were prepared for the cell fusionexperiment.

(6) Cell Fusion

The prepared spleen cells and myeloma cells were mixed and centrifuged(2,000 rpm, 3 minutes). The cells were washed once with 20 ml ofincomplete DMEM and the supernatant was thoroughly removed. Cell fusionwas carried out by grasping the cell vial with hands to maintain atemperature of 37° C. while tapping the lower part of the tube todisrupt the cells. 1 ml of 50% PEG (polyethyleneglycol) solution wasadded dropwise to the tube over 1 minute and the tube was shaken for 90seconds to effect the cell fusion. Exactly 2 minutes and 30 secondsafter adding the first drop of PEG solution, incomplete DMEM was addedto stop the reaction. Here, in order to protect membranes from damagecaused by the osmotic pressure shock upon addition of the PEG solution,the addition of incomplete DMEM was carried out by first adding 1 mlover 1 minute, then 2 ml over 1 minute, then 3 ml over 1 minute and soon, until a total 20 ml of incomplete DMEM had been added to the tube.The Cells thus fused were centrifuged and washed with 20 ml of HATmedium to thoroughly remove PEG. The resulting cells were suspended in20 ml of HAT medium and the resulting solution was added to the 96-wellplates, two drops for each well, and cultured in a CO₂ incubator at 37°C.

On the third day after the cell fusion, three drops of HT medium wereadded to each well. The medium of each well was changed at intervals of3 days and the growth of cells were examined under a microscope.Typically, hybridoma colonies first appeared four days after the fusionand screening of the colonies commenced about 7 days after the fusion.200 μl of the medium was transferred to a 24-well plate containing 400μl of PBS. Cells of wells showing a positive ELISA response weretransferred to a new 24-well plate containing 1 ml of HT medium andcultured for an additional 3 to 4 days. After completion of theculturing, 500 μl of the medium was added to a 15 ml tube containing 2ml of PBS and subjected to a Western blot analysis. Again, cells showinga positive response were transferred to a 6-well plate containing 5 mlof HT medium and cultured. After culturing, the hybridoma cells wereflash frozen and cloned by limiting dilution.

(7) Freezing of Hybridoma Cells

Confluent cells grown in a 10 ml culture flask were centrifuged. Thesettled cells were dissolved in 1 ml of a freezing media containing 90%bovine calf serum, 10% DMSO. The solution was put into a freezer vial,which was placed in a styrofoam box and slowly chilled to −70° C. Aftertwo hours, the vial was quickly transferred to a liquid nitrogen tank,in which the cells can be almost permanently preserved.

(8) Limiting Dilution of Hybridoma Cells

Limiting dilution was carried out to select cells capable of producingantibody against an epitope. Firstly, the number of hybridoma cells inlog phase growth was calculated using a Neubauer Cell Counter andcontinuous dilution was performed until 15 cells were contained in 1 mlmedium, that is, a drop of medium contained one cell. A drop of themedium was added to each well of the 96-well plate containing the feedercells, which had been prepared one or two day(s) ago. Every 3 days, themedium was changed. At 5 days, the plate was scanned using an invertedmicroscope to mark wells where a single colony was observed. At 14 days,hybridoma cells of the marked wells were transferred to a 24-well plateand continuously cultured. After the cultivation, the media were testedby ELISA to identify hybridoma cells producing the desired antibody,which were then stored in a frozen state.

(9) Production of Ascites Fluid

When a large amount of monoclonal antibodies were needed, a BALB/c mousewhich had been injected with 500 μl of pristine 9 days before wasinjected with about 1×10⁷ of the hybridoma cells producing the desiredantibody. 10 to 15 days later, the mouse showing proper abdominaldistension was anesthetized or killed. Ascitic fluid was harvested usingan syringe and centrifuged (15,000 rpm, 10 minutes) to remove cells andtissues. The supernatant was divided into portions, which were stored at−70° C. For subsequent experiments, IgG was isolated from the asciticfluid kept in a frozen state using a Protein A column.

(10) Monoclonal Antibodies for Different Analytes PSA Free PSA AFP CEAAntigen Semen Semen^((a)) Amniotic Human body fluid^((b)) fluid^((c))Captor 32c5 (IgG2a) 83c1 (IgG1) 5c3 (IgG2a) 34 (IgG1) antibody Detector1c1 (IgG2a) 1c1 (IgG2a) 20c4 (IgG1) 17 (IgG2a) antibody CoatingPhosphate Tris Borax Carbonate buffer buffer buffer buffer buffersolution solution solution solution solution (0.1M, (0.15M, (0.2M,(0.5M, pH 7.4) pH 8.0) pH 8.3) pH 9.5) Labeling PBS PBS PBS PBS buffersolution^((a))Obtained from Scripps;^((b))Obtained from RDI;^((c))Obtained from Biodesign

EXAMPLE 2 Preparation of Protein-Fluorescent Material Conjugate

A fluorescent material as a signal generating source was ligated to themouse monoclonal antibody against an analyte of interest for use insubsequent experiments. Proteins to be used in binding of thefluorescent material were purified to a purity of at least 95%. Theproteins were used at a concentration of at least 1 mg/ml for optimalbinding. The purified proteins were dialyzed against a buffer solution(0.1 M sodium bicarbonate, pH 8.5) not containing ammonia or amine ionsin a refrigerator at 4° C. for 12 to 24 hours in order to facilitate thereaction with the fluorescent material. The proteins dialyzed were keptin a refrigerator or −20° C. freezer until use. The proteins dialyzed inthe buffer solution were directly but slowly added to powdered Alexa 647(Molecular Probes, USA) and the reaction was stirred for 1 to 2 hours ina refrigerator at 4° C.

EXAMPLE 3 Purification of Protein-Fluorescent Material Conjugate

Excess unreacted fluorescent material was removed using a distributioncolumn packed with Sephadex G-25. As a purifying buffer solution, 0.1 Msodium carbonate (pH 8.5) was used. The purified protein-fluorescentmaterial conjugates were kept in a refrigerator or −20° C. freezer untiluse.

EXAMPLE 4 Immobilization of Protein on Nitrocellulose Membrane

The proteins were immobilized on a nitrocellulose membrane in a thinline shape with varying the concentration and amount of protein. Themembrane with immobilized proteins was stored in a dehumidifier kept at25° C. and a humidity of 35 to 50% for 2 hours. Then, in order tostabilize the protein and prevent non-specific reactions betweenreagents, the membrane was treated with a stabilizing solution (1% BSA,0.05% Tween 20, 1% sucrose, 0.1% PVA) and equilibrated for 5 minutes. Asthe components of the stabilizing solution, BSA may be substituted withgelatin, Tween 20 may be substituted with Triton X-100, sucrose may besubstituted with trehalose, PVA (polyvinylalcohol) may be substitutedwith PEG or PVP (polyvinylpyrrolidone). After removing excess solutionthe membrane was dried at 40° C. for 30 minutes. The dry membrane wasstored in an appropriate container kept at 25° C. and a humidity of 35to 50% until use.

EXAMPLE 5 Pretreatment of Sample Pad

The sample pad was pretreated in order to facilitate movement ofcomponents of a solution through the nitrocellulose membrane, tomaintain a high sensitivity of reaction and to prevent experimentalerrors due to a non-specific reaction between protein-fluorescentmaterial polymer and a sample.

A sample pad (2.5×30 cm) was sufficiently wetted with a pretreatingsolution (20 mM Tris-Cl, 0.1% Triton X-100, 0.05% NaN₃, pH 8.5) byrepeatedly applying 1 ml of the solution and equilibrating for 10minutes. When whole blood was used as a sample, another pretreatingsolution (PBS, 10 mM phosphate, 150 mM NaCl, 1% BSA, 0.05% Tween 20,0.05% NaN₃, pH 7.4) was used to prevent hemolysis of red blood cells.After removing excess solution, the sample pad was vacuum dried at atemperature of 50° C. to 60° C. for 1 hour to prevent deformation of thepad. The lyophilization method was selected to minimize denaturation ofthe protein-fluorescent material conjugate. The prepared sample wasstored in an appropriate container under the same conditions as for theforegoing membrane.

EXAMPLE 6 Preparation of Conjugate Releasing Pad

The protein-fluorescent material conjugates as a detector for an analyteof interest were immobilized upon a pad made of glass fiber, therebysimplifying the assay procedure to a one step process.

The protein-fluorescent material conjugates were diluted 1/1000, 1/500,1/100 in a buffer solution (PBS, 0.1% gelatin, 0.1% Tween 20, pH 7.4).The general method for applying the mixture to the pad includes soakinga glass fiber pad with the mixture and equilibrating for 5 minutes atroom temperature, followed by drying. However, in this example, themixture was dispensed in an amount of 10, 15, 20 μl/cm using a microdispenser in order to prevent nonuniform redistribution of the mixtureon the surface of the glass fiber pad and to reduce a needed amount ofthe mixture. The conjugate releasing pad (protein-fluorescent materialconjugate pad) can be dried by three methods. The first method was todry the pad at a temperature below 40° C. for 6 hours, consideringstability of the protein component. The second method was to dry the padin a dehumidifier at room temperature for 16 hours. As the third method,lyophilization may be selected to reduce any chance of the proteincomponent being inactivated, though this method requires more time thanthe first method. The prepared conjugate releasing pad was stored in anappropriate container under the same conditions as for the foregoingmembrane.

EXAMPLE 7 Dispensation of Protein on NC (Nitrocellulose) Membrane

Each protein to be immobilized on the membrane was diluted in PBS buffersolution to 1 and 2 mg/ml. The solution was dispensed in an amount of0.88 μl/cm in a line with a width of 0.8 mm on the NC membrane using theBio Dot dispenser and fixed at RH 35 to 50% for 2 hours. Then, themembrane was treated with a stabilizing solution (1% BSA, 0.05% Tween20, 0.1% PVA) for stabilization of proteins and prevention ofnon-specific reactions between reagents, and equilibrated for 5 minutes(As the components of the stabilizing solution, BSA may be substitutedwith gelatin, Tween 20 may be substituted with Triton X-100, sucrose maybe substituted with trehalose, PVA (polyvinylalcohol) may be substitutedwith PEG or PVP (polyvinylpyrrolidone)). After removing excess solution,the treated membrane was dried at 40° C. for 30 minutes. The resultingdry membrane was assembled with the sample pad, absorption pad, etc. andcut to a width of 4 mm using a cutter so that the final strip had adimension of 4×60 mm.

EXAMPLE 8 Quantification of Analyte (Single Test Line)

The captor antibody (1 mg/ml) against PSA (prostate specific antigen) tobe analyzed was dispensed in the test line region on the NC membrane inan amount of 0.88 μl/cm and rabbit IgG (1 mg/ml, 0.1 mg/ml, 0.05 mg/ml,0.01 mg/ml) was dispensed on the reference line in an amount of 0.88μl/cm. The resulting membrane was stored at RH of 35 to 50% for 2 hoursfor immobilization. Then, the membrane was treated with a stabilizingsolution (1% BSA, 0.05% Tween 20, 0.1% PVA) for stabilization ofproteins and prevention of non-specific reactions between reagents, andequilibrated for 5 minutes. After removing excess solution, the treatedmembrane was dried at 40° C. for 30 minutes. The material to react withthe protein to be analyzed via antigen-antibody reaction wasfluorescently-labeled with Alexa 647. Also, the antibody (antigen ?) tobind to the protein dispensed on the reference line via antigen-antibodyreaction was fluorescently-labeled with Alexa 647. Theprotein-fluorescent material conjugates were diluted 1/100 in a dilutionbuffer solution (PBS, 0.1% gelatin, 0.1% Tween 20, pH 7.4). 5% trehaloseas a stabilizing agent was added to the diluted solution. The solutionwas then dispensed in an amount of 20 μl/cm on the surface of glassfiber using a dispenser, followed by lyophilizing.

The prepared NC membrane, conjugate releasing pad, sample pad andabsorption pad were adhered to the backing and assembled in a plastichousing. The PSA standard solution was diluted in a dilution buffersolution (PBST, 10 mM phosphate, 150 mM NaCl, 0.3% Tween 20, pH 7.4) to0, 4, 8, 16 and 32 ng/ml. The concentrations of the prepared standardsolutions were confirmed using a PSA ELISA kit. For proteins dispensedon the reference line, each standard solution as prepared above wasdropped in a specimen input hole of the assay kit and 10 minutes later,the kit was placed in the laser-induced epifluorescence detectingapparatus according to the present invention. The apparatus is designedto express an amount of fluorescence of the detector/analyte/captorconjugates accumulated on a test line or reference line as a peak anddisplay the amount on a monitor. The amount of the Rabbit IgG showing apeak similar to 8 ng/ml PSA was determined as the amount to be dispensedon the reference line. After determining the concentration of thereference line, respective PSA standard solutions were applied to theassay kits while following the same method as described above. 10minutes later, the apparatus displayed the numerical value offluorescence intensity of the analyte which was calculated by inputtinga ratio of the fluorescence intensities of the test line and thereference line into an analogized equation by the polynomial regressionmethod, to obtain the numerical value of the fluorescence intensity ofthe analyte.

EXAMPLE 9 Quantitative Analysis of Total/Free PSA

A monoclonal antibody (1 mg/ml) specifically reacting with total PSA andfree PSA were dispensed on the NC membrane in an amount of 0.88 μl/cm.Separately, a monoclonal antibody having an epitope different from thatof the capture antibody was bound to a fluorescent material, Alexa 647,to form an antibody/fluorescent material conjugate. This conjugate wasmixed with a PBS buffer solution containing 5% trehalose, 1% gelatine asa stabilizer to obtain a 1/100 dilution. A glass fiber pad wasimpregnated with the dilution in an amount of 50 μl/cm², and lyophilizedto obtain a antibody/fluorescent material conjugate pad. The PSAstandard solution was diluted with a dilution buffer solution (PBST, 10mM phosphate, 150 mM NaCl, 0.3% Tween 20, pH 7.4) to 0, 4, 8, 16 and 32ng/ml. The concentrations of the prepared standard solutions wereconfirmed using a PSA ELISA kit. Each standard solution as preparedabove was dropped in a specimen input hole of the assay kit and 15minutes later, each test zone (two zones for total PSA and free PSA) wasexamined for fluorescence intensity using the laser-inducedepifluorescence detecting apparatus according to the present invention.In case of an actual specimen such as serum or whole blood, aconcentration of the specimen was determined using a PSA ELISA kitbefore the specimen was applied to the assay strip. The results areshown in FIG. 19 and FIG. 20.

EXAMPLE 10 Quantitative Analysis of AFP, CEA, PSA, CRP (Multiple TestLines)

A pair of monoclonal angibodies specifically reacting with each ofα-feto protein (AFP, a liver cancer marker), carcinoembryonic antigen(CEA, a tumor marker of many kinds of cancers, mainly used for coloncancer), catabolite regulatory protein (CRP) and PSA was prepared. Thecapture antibodies were dispensed sequentially in test lines atintervals of 2 mm from one end of the chromatography medium on the NCmembrane in an amount of 0.88 μl/cm, one antibody for each test line.Two reference lines were dispensed at 2 mm in front of the first testline and at 2 mm in rear of the last test line, each line being 100ul/ml. The NC membrane with the test and reference lines dispensed wasfixed for 2 hours at RH 35 to 50%. Then, the membrane was treated with astabilizing solution (1% BSA, 0.05% Tween 20, 0.1% PVA) forstabilization of proteins and prevention of non-specific reactionsbetween reagents, and equilibrated for 5 minutes. After removing excesssolution, the treated membrane was dried at 40° C. for 30 minutes. Othermonoclonal antibodies having epitopes different from the immobilizedantibodies were reacted with fluorescent material, to be used asdetectors. Following the same method for quantitative analysis oftotal/free PSA, each conjugate of antibodies and fluorescent materialswas diluted in a dilution buffer solution (5% trehalose, PBS containing0.1% gelatin, pH 7.4) in ratio of 1/100, 1/200, 1/150, and 1/100 forPSA, AFP, CEA and CRP, respectively. A glass fiber pad was impregnatedwith each solution in an amount of 50 μl/cm², followed by lyophilizing,to form a antibody/fluorescent conjugate pad. A specimen, of whichconcentrations had been confirmed using the ELISA kit, was mixed with adilution buffer solution (10 mM phosphate, 150 mM NaCl, 0.3% Tween 20,pH 7.4) and the resulting dilution was applied to the test strip. 10minutes later, intensities of fluorescences from the respective testlines were measured using the laser-induced epifluorescence detectingapparatus according to the present invention. The results are shown inFIG. 16.

EXAMPLE 11 Preparation of Fluorescently-Labeled Antigen or Antibody

Various types of fluorescent material were bound to antibodies andantigens for comparison. FITC (fluorescein-isothiocyanate), rhodamine,Alexa series, Cy3, Cy5 (Molecular probes, Inc.) were used as fluorescentmaterials in this examples. In the experiment, Alexa series, Cy3 and Cy5showed excellent results in stability and reproducibility. In thesubsequent experiments, Alexa 647 was used as a fluorescent material.The prepared fluorescent material/antigen (antibody) conjugate showed astable reactivity and could be used for a sufficiently long period oftime without decoloration.

EXAMPLE 12 Determination of Concentrations of Protein-FluorescentMaterial Conjugate and Immobilized Protein on NC Membrane

In order to determine optimum concentrations of a detector and a captureprotein needed to detect an analyte, serial dilution was performed.Various amounts of a capture protein were immobilized on a NC membrane.Serial dilutions of a protein-fluorescent material conjugate wereprepared. A standard solution of each analyte was mixed with thedilutions of the protein-fluorescent material conjugate and the mixturesolution was applied to a test strip. After the development of thesolution was completed, the test strip was assayed using thelaser-induced epifluorescence detecting apparatus according to thepresent invention. The concentration of the immobilized protein was 1,1.2, 1.4, 1.6, 1.8 and 2 mg/ml for each measurement item and thedispensed amount of the protein was 0.88 μl/cm. At a given concentrationof the capture protein, when the concentration of theprotein-fluorescent material conjugate was increased or decreased, thefluorescence intensity of the analyte at the same concentration was alsoincreased or decreased. Additionally, at a given concentration of theprotein-fluorescent material conjugate, when the concentration of thecapture protein was changed, the same result as above was obtained. Inboth experiments, when the concentration exceeds a certain limit,non-specific reactions increased. By putting the above results together,the optimum concentrations of reagents which can lower the detectionlimit of an analyte and minimize non-specific reactions between thesample and the capture or the detector was determined.

EXAMPLE 13 Minimum Detection Limit of Analyte and Linearity

The PSA monoclonal antibody of the optimum concentration determined inExample 12 was dispensed at amount of 0.88 μl/cm on a NC membrane. Theantibody-fluorescent material conjugate diluted to the concentrationdetermined in Example 12 was mixed with a dilution buffer solution (PBScontaining 5% trehalose, 1% gelatine, pH 7.4). The resulting dilutionwas dispensed to a glass fiber pad in an amount of 20 μl/cm, followed bylyophilization, to obtain a antibody-fluorescent material conjugate pad.Then, the PSA standard solutions at a concentration in a range of 1mg/ml to 1 pg/ml were applied to the strip to determine the minimumdetection limit of the analyte and the linearity range of the assay kitusing the epifluorescence analyser. As shown in FIG. 14, the minimumdetection limit of PSA was 10 pg/ml and the linearity range wasconsiderably wide, from 10 pg/ml to 1 μg/ml. AFP, CEA, CRP of Example 10also showed minimum detection limits much lower than the cut-offsrequired for diagnosis.

EXAMPLE 14

In this example, for comparison, a fluorescence intensity of afluorescent material was measured using the laser-inducedepifluorescence detecting apparatus according to the present inventionand a conventional laser-induced fluorescence detecting scanner, ScanLife, produced by GSI.

Serial dilutions of PSA as an analyte were prepared and mixed with theprotein-fluorescent material. The mixture solution was applied to astrip as in Example 7. The result was imaged using the conventionalscanner. Also, a lateral flow assay strip prepared using the same methodand conditions was imaged using the laser-induced epifluorescencedetecting apparatus according to the present invention. The imaged datawere converted into numerical data using a related program. The resultsare shown in FIG. 15. From the results of FIG. 15, it was noted thatboth the laser-induced epifluorescence detecting apparatus according tothe present invention and the conventional fluorescence detectingscanner showed a fluorescence intensity increasing according to theconcentration of the analyte, while the fluorescence intensity measuredby the laser-induced epifluorescence detecting apparatus according tothe present invention was much higher than that measured by theconventional fluorescence detecting scanner.

EXAMPLE 15 Determination of Location of Reference Line

In order to determine the location of a reference line, three types ofstrips having a reference line in front of the test line, at the rear ofthe test line, and two reference lines at both locations, were prepared.For each type of strip, an analyte at different concentrations wasassayed, each concentration being assayed 10 times. Thus, each of theanalyte standard solutions at different concentrations was mixed with aprotein-fluorescent material conjugate and applied to the test strip,followed by analysis by the laser-induced fluorescence detectingapparatus. The results are shown in Table 5 below, in which thedifferences between the strips are given as error ranges of the analyzedvalues. Although the error range of each strip was not out of theacceptable error range, when the reference line was located in front ofthe test line, or in front of the test line and at the rear of the testline, accuracy and reproducibility were preferably higher.

Accordingly, it was concluded that for reliable and reproducibleresults, the reference line should be located in front of the test lineor both in front and at the rear of the test line, though the results inany position was not out of the acceptable error range. TABLE 5 40 pg/ml400 pg/ml 4 ng/ml Front 40.2 ± 4 403.6 ± 12 4.01 ± 0.2 Rear 41.3 ± 6405.2 ± 35 4.09 ± 0.4 Front and rear 40.2 ± 3 403.6 ± 11 4.01 ± 0.1

EXAMPLE 16 Dispensation of Avidin Protein on NC (Nitrocellulose)Membrane

Avidin to be immobilized was diluted in PBS buffer solution to 1 or 2mg/ml. The solution was dispensed in an amount of 0.88 μl/cm as a linewith a width of 0.8 mm on the NC membrane using the Bio Dot dispenserand fixed at RH 35 to 50% for 2 hours. Then, the membrane was treatedwith a stabilizing solution (1% BSA, 0.05% Tween 20, 0.1% PVA) forstabilization of proteins and prevention of non-specific reactionsbetween reagents, and equilibrated for 5 minutes (As the components ofthe stabilizing solution, BSA may be substituted with gelatin, Tween 20may be substituted with Triton X-100, sucrose may be substituted withtrehalose, PVA (polyvinylalcohol) may be substituted with PEG or PVP(polyvinylpyrrolidone)). After removing excess solution, the treatedmembrane was dried at 40° C. for 30 minutes. The resulting dry membranewas assembled with the sample pad, absorption pad, etc. and cut to awidth of 4 mm using a cutter so that the final strip had a dimension of4×60 mm.

EXAMPLE 17 Quantification of Analyte Using Avidin-Biotin (Single TestLine)

The avidin (1 mg/ml) and rabbit IgG (1 mg/ml) were dispensed in the testline region and reference line in an amount of 0.88 μl/cm over the NCmembrane. The resulting membrane was stored at RH of 35 to 50% for 2hours for immobilization. Then, the membrane was treated with astabilizing solution (1% BSA, 0.05% Tween 20, 0.1% PVA) forstabilization of proteins and prevention of non-specific reactionsbetween reagents, and equilibrated for 5 minutes. After removing excesssolution, the treated membrane was dried at 40° C. for 30 minutes. Thematerial to react with the protein to be analyzed via antigen-antibodyreaction was fluorescently-labeled with Alexa 647. The protein tocapture the analyte was coupled with biotin. Also, the antibody to bindto the protein dispensed on the reference line via antigen-antibodyreaction was fluorescently-labeled with Alexa 647. Theprotein-fluorescent material conjugate and the protein-biotin conjugatewere diluted 1/100 in a dilution buffer solution (PBS, 0.1% gelatin,0.1% Tween 20, pH 7.4).

The prepared NC membrane, sample pad and absorption pad were adhered tothe backing, cut to a dimension of 4×60 mm and assembled in a plastichousing. The PSA standard solution was diluted in a dilution buffersolution (PBST, 10 mM phosphate, 150 mM NaCl, 0.3% Tween 20, pH 7.4) to0, 4, 8, 20 and 40 ng/ml. The concentrations of the prepared standardsolutions were confirmed using a PSA ELISA kit. Each of the prepared PSAstandard solutions, protein-Alexa 647 conjugate and captureprotein-biotin, and the protein-Alexa 647 capable of recognizing theprotein dispensed on the reference line, were dropped in a specimeninput hole of the assay kit and 10 minutes later, the kit was placed inthe laser-induced epifluorescence detecting apparatus according to thepresent invention. The apparatus was designed to display thefluorescence intensity of the detector/analyte/capture conjugateaccumulated on the test line and fluorescence intensity ofprotein-fluorescent material conjugate on the reference line as peaks ona monitor. Also, the apparatus displayed the numerical value offluorescence intensity of the analyte which was calculated by inputtinga ratio of the fluorescence intensities of the test line and thereference line into an analogized equation by the polynomial regressionmethod, to obtain the numerical value of the fluorescence intensity ofthe analyte. The results are shown in FIG. 17. The line A represents theresult of the quantitative assay using avidin-biotin and the line Brepresents the result of the quantitative assay according to theconventional method without using avidin-biotin as a control. From theresults of FIG. 17, it is noted that greater sensitivity andreproducibility can be obtained by using avidin-biotin.

EXAMPLE 17

A protein chip was prepared using polystyrene as a substrate. Each ofthe proteins prepared in Example 1 was mixed with 50 mM sodiumbicarbonate (pH 9.6), 20 mM Tris-Cl (pH 8.5), 10 mM PBS (pH 7.2). Themixture was dispensed on the substrate, followed by immobilization atroom temperature for 2 hours. After completion of the reaction, thesubstrate was washed twice with distilled water and a blocking buffersolution (PBS containing 0.1% BSA, 0.05% Tween 20) was added to thesubstrate, followed leaving at room temperature for 10 minutes, toobtain a protein chip.

Using the laser-induced epifluorescence detecting apparatus according tothe present invention, constructed as shown in FIG. 2, the fluorescencesemitted by the protein where the antibody proteins prepared as abovewere arranged in one dimension were measured. The results are shown inFIG. 22.

INDUSTRIAL APPLICABILITY

As described above, the lateral flow assay strip according to thepresent invention makes it possible to measure analyte(s) at the sametime, with high sensitivity. In addition, a laser-inducedepifluorescence detector of the present invention provides a collectionefficiency of at least 80% fluorescence and thus makes it possible tomeasure multiple analytes by minimum detection limit of pg/ml.

1. A method for detecting laser-induced epifluorescence which comprisessteps of: passing light emitted from a laser through an exciter filterand focusing the filtered light to the surface of a sample positioned ata first focal point of an elliptical or spherical reflecting mirror witha proper size, wherein the sample is positioned by an epifluorescentsample control means; focusing scattered incident light and fluorescenceemitted from the sample positioned at the first focal point of theelliptical reflecting mirror to a second focal point of the ellipticalreflecting mirror by reflection of the reflecting mirror; converting thefocused light into parallel light by a collimator; filtering theparallel light through a fluorescent filter to remove the scatteredincident light; and providing a pure a fluorescence component to anoptical detector.
 2. The method of claim 1, wherein the scatteredincident light and fluorescence reflected by the elliptical reflectingmirror are focused to a spatial filter positioned, and thereafter,enters the collimator.
 3. A laser-induced epifluorescence detectingapparatus comprising a laser, an exciter filter, an ellipticalreflecting mirror or spherical reflecting mirror, epifluorescent samplecontrol means, a collimator, a fluorescent filter and an opticaldetector, characterized in that the components of the detectingapparatus are arranged in a structure such that light emitted from alaser is passed through an exciter filter and is focused to the surfaceof a sample positioned at a first focal point of an ellipticalreflecting mirror, scattered incident light and fluorescence emittedfrom the sample positioned at the first focal point of the ellipticalreflecting mirror are reflected from the elliptical reflecting mirrorand focused to a collimator as a second focal point of the ellipticalreflecting mirror, by which the focused light is converted into parallellight and passed through a fluorescent filter to remove the scatteredincident light, thereby providing a pure fluorescence component to anoptical detector.
 4. The apparatus of claim 3, wherein the scatteredincident light and fluorescence reflected by the elliptical reflectingmirror are focused upon a spatial filter positioned, and thereafter,enters the collimator.
 5. The apparatus of claim 3, wherein the sampleis be moved upward and downward or back and forth by the sample controlmeans.
 6. The apparatus of claim 4, wherein the sample is be movedupward and downward or back and forth by the sample control means.